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

Inha University - Bioengineering

• AbstractThe 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.

• AbstractThe 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.

Inha University - Bioengineering

Presentation contains

Introduction

Materials and Methods

Result

Discussion

Presentation contains

Introduction

Materials and Methods

Result

Discussion

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.

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

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

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

Inha University - Bioengineering

Results

Effect of pH on melanin synthesis in humans FM94 melanoma cell lysate

AVisualization 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)

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)

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.

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.

Thank You