Ann Dermatol.  2012 May;24(2):151-157.

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

• Hah YS
• Cho HY
• Lim TY
• Park DH
• Kim HM
• Yoon J
• Kim JG
• Kim CY
• Yoon TJ

Abstract

BACKGROUND
Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis.
OBJECTIVE
The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of alpha-melanocyte-stimulating hormone (alpha-MSH) were evaluated.
METHODS
In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot.
RESULTS
In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and alpha-MSH increased melanogenesis more strongly than alpha-MSH alone.
CONCLUSION
Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with alpha-MSH in enhancing melanogenesis in melanocyte cells.

Keyword

Melanin synthesis; Melanoma; mTOR; Rapamycin; Tyrosinase

MeSH Terms

alpha-MSH
Androstadienes
Animals
Cell Survival
Chromones
Humans
Melanins
Melanocytes
Melanoma
Mice
Microphthalmia-Associated Transcription Factor
Monophenol Monooxygenase
Morpholines
Phosphatidylinositol 3-Kinase
Sirolimus
Up-Regulation
Androstadienes
Chromones
Melanins
Microphthalmia-Associated Transcription Factor
Monophenol Monooxygenase
Morpholines
Phosphatidylinositol 3-Kinase
Sirolimus
alpha-MSH

Figure

• 

  Fig. 1 Effects of rapamycin on melanin content and the proliferation of MNT-1 cells. MNT-1 cells were treated with various concentrations of rapamycin for 48 hours. (A) Melanin content. (B) Cell viability determined by the metabolism of 3-(4, 5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Melanin content and cell proliferation are expressed as percentage of the control. Data represent means±standard deviation of at least three independent experiments. Significant differences were determined by Student's t-test; *p<0.01, **p<0.001 versus control.

• 

  Fig. 2 Effects of rapamycin on Tyrosinase (TYR) activity in MNT-1 cells. MNT-1 cells were treated with rapamycin (100 nM) or α-melanocyte-stimulating hormone (α-MSH) (100 nM) for the indicated times. (A) Melanin content. (B) TYR activity determined by measuring the formation of dopachrome. Melanin content and TYR activity are expressed as percentage of the control. Data are expressed as means±standard deviation of at least three independent experiments. *p<0.01, **p<0.001 versus control.

• 

  Fig. 3 Effects of rapamycin on expression of Tyrosinase (TYR), TYRP-1, TYRP-2 and MITF proteins. (A) MNT-1 cells were treated with various concentrations of rapamycin (10, 100, 1,000 nM) and α-melanocyte-stimulating hormone (100 nM) as noted for the indicated times. Cells were then subjected to Western blot analysis performed using antibodies against TYR, TYRP-1, TYRP-2 and MITF. Equal protein loadings were confirmed using an anti-actin antibody. (B) The amount of protein (arbitrary unit) was quantified using an imaging densitometer. The results are averages of triplicate experiments, and the data are expressed as mean±standard deviation. Rm: Rapamycin, MSH: α-melanocyte-stimulating hormone, TYRP: tyrosinase-related protein, MITF: microphthalmia-associated transcription factor.

• 

  Fig. 4 Effects on melanin production. MNT-1 cells were stimulated with 100 nM rapamycin (Rm) and/or 1 µM wortmannin (Wm) and/or 100 nM α-melanocyte-stimulating hormone (MSH) for 72 hours. (A) Macroscopic view of cell pellets. (B) After the incubation period, melanin contents were measured and are expressed as percentage of the untreated control (mean±standard deviation). *p<0.05, **p<0.001 versus control.

Reference

1. Hearing VJ, Jiménez M. Analysis of mammalian pigmentation at the molecular level. Pigment Cell Res. 1989. 2:75–85.

2. Buscà R, Ballotti R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res. 2000. 13:60–69.

3. Bertolotto C, Abbe P, Hemesath TJ, Bille K, Fisher DE, Ortonne JP, et al. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes. J Cell Biol. 1998. 142:827–835.

4. Oka M, Ogita K, Saito N, Mishima Y. Selective increase of the alpha subspecies of protein kinase C and inhibition of melanogenesis induced by retinoic acid in melanoma cells. J Invest Dermatol. 1993. 100:2 Suppl. 204S–208S.

5. Park HY, Russakovsky V, Ohno S, Gilchrest BA. The beta isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells. J Biol Chem. 1993. 268:11742–11749.

6. Englaro W, Rezzonico R, Durand-Clément M, Lallemand D, Ortonne JP, Ballotti R. Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells. J Biol Chem. 1995. 270:24315–24320.

7. Tada A, Pereira E, Beitner-Johnson D, Kavanagh R, Abdel-Malek ZA. Mitogen- and ultraviolet-B-induced signaling pathways in normal human melanocytes. J Invest Dermatol. 2002. 118:316–322.

8. Wong G, Pawelek J. Melanocyte-stimulating hormone promotes activation of pre-existing tyrosinase molecules in Cloudman S91 melanoma cells. Nature. 1975. 255:644–646.

9. Halaban R, Pomerantz SH, Marshall S, Lerner AB. Tyrosinase activity and abundance in Cloudman melanoma cells. Arch Biochem Biophys. 1984. 230:383–387.

10. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006. 124:471–484.

11. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004. 306:990–995.

12. Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr Opin Cell Biol. 2005. 17:596–603.

13. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 2006. 441:424–430.

14. Ohguchi K, Banno Y, Nakagawa Y, Akao Y, Nozawa Y. Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. J Cell Physiol. 2005. 205:444–451.

15. Buscà R, Bertolotto C, Ortonne JP, Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70(S6)-kinase pathway induces B16 melanoma cell differentiation. J Biol Chem. 1996. 271:31824–31830.

16. Oka M, Nagai H, Ando H, Fukunaga M, Matsumura M, Araki K, et al. Regulation of melanogenesis through phosphatidylinositol 3-kinase-Akt pathway in human G361 melanoma cells. J Invest Dermatol. 2000. 115:699–703.

17. Choi TY, Kim JH, Ko DH, Kim CH, Hwang JS, Ahn S, et al. Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pigment Cell Res. 2007. 20:120–127.

18. Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC Jr, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996. 15:5256–5267.

19. Hartley KO, Gell D, Smith GC, Zhang H, Divecha N, Connelly MA, et al. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995. 82:849–856.

20. Ui M, Okada T, Hazeki K, Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci. 1995. 20:303–307.

21. Hunt G, Todd C, Cresswell JE, Thody AJ. Alpha-melanocyte stimulating hormone and its analogue Nle4DPhe7 alpha-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes. J Cell Sci. 1994. 107:205–211.

22. Ando H, Itoh A, Mishima Y, Ichihashi M. Correlation between the number of melanosomes, tyrosinase mRNA levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agents. J Cell Physiol. 1995. 163:608–614.

23. Körner A, Pawelek J. Activation of melanoma tyrosinase by a cyclic AMP-dependent protein kinase in a cell-free system. Nature. 1977. 267:444–447.

24. Kimura K, Hattori S, Kabuyama Y, Shizawa Y, Takayanagi J, Nakamura S, et al. Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1994. 269:18961–18967.