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Molecular and Cellular Biochemistry

, Volume 415, Issue 1–2, pp 103–109 | Cite as

The transcription factor TBX2 regulates melanogenesis in melanocytes by repressing Oca2

  • Yu Chen
  • Li Pan
  • Zhongyuan Su
  • Jing Wang
  • Huirong Li
  • Xiaoyin Ma
  • Yin Liu
  • Fan Lu
  • Jia Qu
  • Ling HouEmail author
Article

Abstract

The T-box transcription factor TBX2 is known for its role as a critical regulator of melanoma cell proliferation, but its role in regulating melanogenesis has not been widely studied. Here we use a series of experiments to show in primary and immortalized mouse melanocytes that TBX2 acts as regulator of melanogenesis by repressing the expression of the gene encoding the melanosomal protein OCA2. We find that α-MSH or forskolin, both of which stimulate melanogenesis, also reduce TBX2 expression, and that specific knockdown of TBX2 increases melanogenesis. This effect primarily involves an increase in Oca2 expression as the combined knockdown of both Tbx2 and Oca2 interferes with the Tbx2 knockdown-mediated increase in melanogenesis. Standard chromatin immunoprecipitation and reporter assays suggest that TBX2 represses Oca2 at least in part directly. Hence, the results suggest that TBX2 may act as a nexus linking cell proliferation and melanogenesis.

Keywords

T-box transcription factor Transcriptional regulation Pigmentary disease Melanocyte Pigmentation 

Notes

Acknowledgments

We thank Dr. Dorothy Bennett for reagents, and Dr. Heinz Arnheiter for thoughtful comments on the manuscript. This work was supported by the National Natural Science Foundation of China (81570892, 31201031), the Natural Science Foundation of Zhejiang Province (LQ16C070001, LQ13H120004,LQ13H120004,LY13C090004), and the Research Grant of Wenzhou Medical University.

Supplementary material

11010_2016_2680_MOESM1_ESM.jpg (861 kb)
Figure S1. α-MSH or forskolin promotes melanogenesis in primary melanocytes. After treatment with 100 nM α-MSH or 5μM forskolin for three days, we took bright-field images of primary melanocytes (A) and analyzed melanin content (B). Black arrows indicate heavily pigmented melanocytes. (C) After treatment with 100nM α-MSH or 5μM forskolin for two days, the expression levels of several melanogenesis-associated genes were examined by relative real-time PCR. Note that in primary melanocytes, the expression of Tbx2 was downregulated after treatment with α-MSH or 5μM forskolin. Experiments were done in triplicates and error bars were represented as mean ±SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Scale bar is 50 μm. Supplementary material 1 (JPEG 861 kb)
11010_2016_2680_MOESM2_ESM.jpg (1.2 mb)
Figure S2. Knockdown of Tbx2 promotes melanogenesis in primary melanocytes and melan-a cells. (A-B) 4 days after transfection with si-C, si-Tbx2-1 or si-Tbx2-2, bright-field images of primary melanocytes were recorded (A) and melanin content was analyzed (B). (C-E) Four days after transfection with si-C, si-Tbx2-1 or si-Tbx2-2, transmitted bright-field images of melan-a cells were recorded (C), pellets of melan-a cells were collected (D), and melanin content was analyzed (E). Black arrows indicate heavily pigmented melanocytes. Note that pigmentation was increased after knockdown of Tbx2. Experiments were done in triplicates and error bars were represented as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Scale bar is 50 μm. Supplementary material 2 (JPEG 1272 kb)
11010_2016_2680_MOESM3_ESM.doc (27 kb)
Supplementary material 4 (DOC 27 kb)

References

  1. 1.
    Slominski A, Tobin DJ, Shibahara S, Wortsman J (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155–1228CrossRefPubMedGoogle Scholar
  2. 2.
    Slominski A, Zmijewski MA, Pawelek J (2012) l-tyrosine and l-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions. Pigment Cell Melanoma Res 25:14–27CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hou L, Pavan WJ (2008) Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development: do all roads lead to Mitf? Cell Res 18:1163–1176CrossRefPubMedGoogle Scholar
  4. 4.
    Hacker SM (1996) Common disorders of pigmentation: when are more than cosmetic cover-ups required? Postgrad Med 99:177–186PubMedGoogle Scholar
  5. 5.
    Carreira S, Liu B, Goding CR (2000) The gene encoding the T-box factor Tbx2 is a target for the microphthalmia-associated transcription factor in melanocytes. J Biol Chem 275:21920–21927CrossRefPubMedGoogle Scholar
  6. 6.
    Liu F, Cao J, Lv J, Dong L, Pier E, Xu GX, Wang RA, Xu Z, Goding C, Cui R (2013) TBX2 expression is regulated by PAX3 in the melanocyte lineage. Pigment Cell Melanoma Res 26:67–77CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Abrahams A, Parker MI, Prince S (2010) The T-box transcription factor Tbx2: its role in development and possible implication in cancer. IUBMB Life 62:92–102PubMedGoogle Scholar
  8. 8.
    Vance KW, Carreira S, Brosch G, Goding CR (2005) Tbx2 is overexpressed and plays an important role in maintaining proliferation and suppression of senescence in melanomas. Cancer Res 65:2260–2268CrossRefPubMedGoogle Scholar
  9. 9.
    Pan L, Ma X, Wen B, Su Z, Zheng X, Liu Y, Li H, Chen Y, Wang J, Lu F, Qu J, Hou L (2015) Microphthalmia-associated transcription factor/T-box factor-2 axis acts through cyclin D1 to regulate melanocyte proliferation. Cell Prolif 48:631–642CrossRefPubMedGoogle Scholar
  10. 10.
    Carreira S, Dexter TJ, Yavuzer U, Easty DJ, Goding CR (1998) Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter. Mol Cell Biol 18:5099–5108CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Brilliant MH (2001) The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH. Pigment Cell Res 14:86–93CrossRefPubMedGoogle Scholar
  12. 12.
    Bellono NW, Escobar IE, Lefkovith AJ, Marks MS, Oancea E (2014) An intracellular anion channel critical for pigmentation. Elife 16:e04543Google Scholar
  13. 13.
    Chen K, Minwalla L, Ni L, Orlow SJ (2004) Correction of defective early tyrosinase processing by bafilomycin A1 and monensin in pink-eyed dilution melanocytes. Pigment Cell Res 17:36–42CrossRefPubMedGoogle Scholar
  14. 14.
    Cheng T, Orlow SJ, Manga P (2013) Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes. Pigment Cell Melanoma Res 26:826–834CrossRefPubMedGoogle Scholar
  15. 15.
    Toyofuku K, Valencia JC, Kushimoto T, Costin GE, Virador VM, Vieira WD, Ferrans VJ, Hearing VJ (2002) The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase. Pigment Cell Res 15:217–224CrossRefPubMedGoogle Scholar
  16. 16.
    Gardner JM, Nakatsu Y, Gondo Y, Lee S, Lyon MF, King RA, Brilliant MH (1992) The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes. Science 257:1121–1124CrossRefPubMedGoogle Scholar
  17. 17.
    Rosemblat S, Durham-Pierre D, Gardner JM, Nakatsu Y, Brilliant MH, Orlow SJ (1994) Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene. Proc Natl Acad Sci USA 91:12071–12075CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Martinez-Garcia M, Montoliu L (2013) Albinism in Europe. J Dermatol 40:319–324CrossRefPubMedGoogle Scholar
  19. 19.
    Bennett DC, Cooper PJ, Hart IR (1987) A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth. Int J Cancer 39:414–418CrossRefPubMedGoogle Scholar
  20. 20.
    Manning CS, Hooper S, Sahai EA (2015) Intravital imaging of SRF and Notch signalling identifies a key role for EZH2 in invasive melanoma cells. Oncogene 34:4320–4332CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kim JY, Shin JY, Kim MR, Hann SK, Oh SH (2012) siRNA-mediated knock-down of COX-2 in melanocytes suppresses melanogenesis. Exp Dermatol 21:420–425CrossRefPubMedGoogle Scholar
  22. 22.
    Ludtke TH, Farin HF, Rudat C, Schuster-Gossler K, Petry M, Barnett P, Christoffels VM, Kispert A (2013) Tbx2 controls lung growth by direct repression of the cell cycle inhibitor genes Cdkn1a and Cdkn1b. PLoS Genet 9:e1003189CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Paxton C, Zhao H, Chin Y, Langner K, Reecy J (2002) Murine Tbx2 contains domains that activate and repress gene transcription. Gene 283:117–124CrossRefPubMedGoogle Scholar
  24. 24.
    Ismland F, McGowan K, Rubin CJ, Henegar C, Sundström E, Berglund J, Schwochow D, Gustafson U, Imsland P, Lindblad-Toh K, Lindgren G, Mikko S, Millon L, Wade C, Schubert M, Orlando L, Penedo MC, Barsh GS, Andersson L (2016) Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses. Nat Genet 48(2):152–158CrossRefGoogle Scholar
  25. 25.
    Harrelson Z, Kelly RG, Goldin SN, Gibson-Brown JJ, Bollag RJ, Silver LM, Papaioannou VE (2004) Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 131:5041–5052CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yu Chen
    • 1
  • Li Pan
    • 1
  • Zhongyuan Su
    • 1
  • Jing Wang
    • 1
  • Huirong Li
    • 1
  • Xiaoyin Ma
    • 1
  • Yin Liu
    • 1
  • Fan Lu
    • 2
  • Jia Qu
    • 2
  • Ling Hou
    • 1
    • 2
    Email author
  1. 1.Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye HospitalWenzhou Medical UniversityWenzhouChina
  2. 2.State Key Laboratory Cultivation Base and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of OphthalmologyWenzhou Medical UniversityWenzhouChina

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