1. Inha University - Bioengineering
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*,1
*Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, United Kingdom; †Department of
Dermatology, Leiden
University Medical Center (LUMC), The Netherlands; and ‡Fujita Health University, School of Health Sciences, Japan
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*,1
*Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, United Kingdom; †Department of
Dermatology, Leiden
University Medical Center (LUMC), The Netherlands; and ‡Fujita Health University, School of Health Sciences, Japan

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 melanogenesis. 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.
• 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 melanogenesis. 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
Presentation contains
Introduction
Materials and Methods
Result
Discussion
Presentation contains
Introduction
Materials and Methods
Result
Discussion

4. Inha University -
Bioengineering
• Introduction
 Why skin color varies between races and between individual of same ethnic color?
 Why copper containing metallo-enzyme tyrosinase is considered as a rate limiting enzyme?
 several studies have shown that cells with similar amounts of tyrosinase still display differences in tyrosinase
activity and hence melanogenesis.
 Melanin production takes place within specialized intracellular organelles known as melanosomes. There is
evidence that melanosomes are closely related to lysosomes. For instance, both organelles contain the same
structural proteins (e.g., LAMP, acidic hydrolyses, vacuolar type proton pumps) and both are affected in genetic
disorders such as the Chediak–Higashi and Hermansky–Pudlak syndrome.
 Direct measurements have shown that melanosomes can be acidic. Thus it has been suggested that the
melanosome represents a highly specialized lysosome rather than a completely unique structure.
 Ammonium chloride facilitate maturation of melonosomes. Hence the factor that regulate maturation rate of
melanosomes might be expected to affect melanin distribution in epidermis and skin pigmenttation.
 The aim of the present study was to further test this hypothesis and to expand it to different human skin types.
We have examined the effects of increasing melanosomal pH on tyrosinaseactivity, melanogenesis, melanosome
maturation, and eumelanin/phaeomelanin ratio in normal human melanocytes (NHM) from the whole of range
human skin types (I–VI) as well as melanoma cells showing different levels of melanogenesis.
• Introduction
 Why skin color varies between races and between individual of same ethnic color?
 Why copper containing metallo-enzyme tyrosinase is considered as a rate limiting enzyme?
 several studies have shown that cells with similar amounts of tyrosinase still display differences in tyrosinase
activity and hence melanogenesis.
 Melanin production takes place within specialized intracellular organelles known as melanosomes. There is
evidence that melanosomes are closely related to lysosomes. For instance, both organelles contain the same
structural proteins (e.g., LAMP, acidic hydrolyses, vacuolar type proton pumps) and both are affected in genetic
disorders such as the Chediak–Higashi and Hermansky–Pudlak syndrome.
 Direct measurements have shown that melanosomes can be acidic. Thus it has been suggested that the
melanosome represents a highly specialized lysosome rather than a completely unique structure.
 Ammonium chloride facilitate maturation of melonosomes. Hence the factor that regulate maturation rate of
melanosomes might be expected to affect melanin distribution in epidermis and skin pigmenttation.
 The aim of the present study was to further test this hypothesis and to expand it to different human skin types.
We have examined the effects of increasing melanosomal pH on tyrosinaseactivity, melanogenesis, melanosome
maturation, and eumelanin/phaeomelanin ratio in normal human melanocytes (NHM) from the whole of range
human skin types (I–VI) as well as melanoma cells showing different levels of melanogenesis.

5. Inha University - Bioengineering
• Cell and cell cultures
 11 human melanocyte and 3 melanoma cell lines
 Melanoma cells (RPMI) (10% FBS)
 Human melanocyte (Ham’s F10) (16 nM TPA, 2.5 nM Cholera toxin, 0.1 mM IBMX and 1%
UltrosgerG (Gibco BRL)
 For melanogenesis assay medium was supplemented with 1mM L-tyrosine.
 Selective vacuolar type H+ ATPase inhibitor bafilomycin (BafA1) and concanamycin (ConA)
(Sigma and calbiochem)
RT-PCR
 RNA (total) Tri Reagent (Sigma)
 Dynabids
 cDNA – MoMLV reverse transcription kit (Frementas)
 Primers
 Cycling Parameters (initially 10 cycle of 94-30S, 35- 30S (Decrease0.5 per cycle) 72-1Min followed
by 25 cycle of annealing at 60.
Materials and methods

6. Inha University - Bioengineering
• Tyrosinase Activity
• Melanin Assay
-The amount of melanin was assayed by dissolving washed pellet directly in 1
ml soluene 350
-Absorbance – 475nM
-Protein concentration, Lowry assay kit (BioRad)
• Labelling of cells with Acridine orange (AO)
-20 mM AO
-BafA1 and ConA
-The cells then examined using a Leica DMRXA microscope. (Equipped with cooled CCD camera)
-The green and red fluorescence were recorded using (BP353/50) and
BP610/80) filters and image combined using color Proc 98 software
Materials and methods

7. Inha University - Bioengineering
• Measurement of Eumelanin and Pheomelanin
– Involves paramagnetic oxidation of eumelanin to pyrole-2,3-5 tricarboxyllic acid (PTCA) and the
hydriodic acid hydrolysis of pheomelani to aminohydroxyphenylalanin (AHP)
• Light and Transmission Electron Microscopy
Materials and methods

8. Inha University - Bioengineering
Results
Effect of pH on melanin synthesis in
humans FM94 melanoma cell lysate
A
Visualization of acidic organelle . Red
Fluorescence indicates the presence of
acidic visicles under control conditions in
C803 )A) FM55 (C), and FM94( E).
Treatment with ConA (10 nM) caused
neutralisation of acidic organelles in all
cll cultures tested as represented as B,D,F
similar effects were senn with
BafA1(20nM)

9. Inha University - Bioengineering
Effect of 24 hours incubation with proton pump inhibitor ConA (10nM) on tyrosinase acyivity (A and B) and melanin content and type (C and D). Tyrosinase activity was
measured for the cells with low (A) and medium high basal activity (B) incubated in the absence (striped bars) or presence of proton pump inhibitors (closed bars). Total
melanin content © and eumelanin to pheomelanin ratio represented as PTCA/AHP (D) was measured for representatives cultures and expressed as the percentage increase
in response to proton pump inhibitor . (Similr effects were seen with BafA1 (20nM)

10. Inha University - Bioengineering
Effects of ConA (10nM) on the numbers of mature melanosomes in C36 melanocyte as reveled using thin light microscopy (A,B) and EM (C,D). After 24 hours of
treatment with proton pump inhibitor increases in pigment granules numbers were observed (B and D) compared with controls (A and C). Arrow points the mature
melanosomes.

11. Inha University - Bioengineering
MSh
Discussion
Melanosomal pH is controlled and maintained by the V-type proton
pump and p protein. The abundance, polymorphism and relative
activity of these proteins could serve as key controls points for
melanosomal pH. Modulation of melanosomal pH affects activity of
tyrosinase. The ratio of eumelanin/pheomelanin production, and
maturation rate of the melanosome. It is possible that UVR and alpha
MSH stimulate eumelanogenesis by neutralisation of melanosomal pH
through an increase in p protein expression.
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