130427 paper study 준섭

Melanosomal pH Controls Rate of Melanogenesis, Eumelanin/Phaeome-lanin Ratio and Melanosome Matura-tion in Melanocytes and Melanoma

CellsJanis 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

INHA University - Bioengineer-ingABSTRACT

The skin pigment melanin is produced in melanocytes in highly specialized or-ganelles 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 ef-fect 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 re-sponse to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy re-vealed 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 polymor-phism.


<me-lanocyte>

melanosome

endosomelysosomepH ↓↓

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,


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

INHA University - Bioengineer-ingINTRODUCTION

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 ac-tivity 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 (yel-low to reddish brown).

•races•same ethnic group skin color

varies

Why??

melanosome 의 pH 가 다르다 !

INHA University - Bioengineer-ingMATERIALS 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 Phaeome-laninLight and Transmission Electron Mi-croscopy

Human melanoma cell line :FM3, FM55, FM81, FM94

INHA University - Bioengineer-ingRESULTS

Characterization of Melanogenic System in Human Pigment CellsCharacterization of Melanogenic System in Human Pigment Cells

FM94 melanoma cell

optimal pH near neutral pH


Effect of Neutralizing Melanosomal pH on Melanogenesis and Eumelanin/Phaeomelanin Ratio


FIG. 2. Visualization of acidic or-ganelles.

Red fluorescence indicates the presence of acidic vesi-cles

(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




<tyrosinase as-say>

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)




FIG. 3. Effects of 24 h incubation with proton pump inhibitor ConA (10 nM) on melanin content and type (C and D)

CellConA treat •increase of melanin

•increase of PTCA/AHP ra-

tio※PTCA : eumelanin※AHP : phaeome-lanin


Effects of Neutralizing Melanosomal pH on Melanosome MaturationEffects 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). Af-ter 24 h of treatment with proton pump inhibitor increases in pigment granule num-bers were observed (B and D) compared with controls (A and C). Arrows point to ma-ture melanosomes; scale bars 5 5 mm (A, B) and 1 mm (C, D).

INHA University - Bioengineer-ingDISCUISSION

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

130427 paper study 준섭

Technology