Different cis-Acting Elements Are Involved in the Regulation of TRP1 and TRP2 Promoter Activities by Cyclic AMP: Pivotal Role of M Boxes (GTCATGTGCT) and of Microphthalmia

Abstract

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.In mammals, pigmentation results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. Melanin synthesis (melanogenesis) takes place in the melanocyte after differentiation of the nonpigmented precursor, the melanoblast (27). Three melanocyte-specific enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2, are involved in this enzymatic process that converts tyrosine to melanin pigments. Although these proteins have similar structures and features, they are expressed by different genes and possess distinct enzymatic activities. Tyrosinase, encoded by the albino locus of the mouse, catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and of DOPA to DOPA quinone (14,25,31). TRP2, encoded by the mouse slaty locus, possesses a Dopachrome tautomerase activity, converting the Dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (3,19,42). TRP1, which has been mapped in mouse to the brown locus, catalyzes the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid (21, 24).In vivo, melanogenesis is regulated by UVB radiation that can act either directly on melanocytes or indirectly through the release of keratinocyte-derived factors such as interleukins, prostaglandins, and alpha melanocyte-stimulating hormone (␣-MSH) (1,12,22,35). Interestingly, ␣-MSH, one of the most potent activators of melanogenesis, binds to an ␣ s -coupled rece...

Cyclic AMP a Key Messenger in the Regulation of Skin Pigmentation

“…Experiments using a 2.2 kb fragment of the mouse tyrosinase promoter cloned upstream of the luciferase reporter gene, have revealed that this promoter activity is strongly activated by a-MSH, cAMP elevating agents and by the over-expression of the catalytic subunit of PKA (46,47). However, the sequence analysis of this promoter has not revealed the existence of any classical CRE site, which means that PKA does not directly regulate tyrosinase expression through CREB activation.…”

[6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

“…Tyrosinase regulates two catalytic processes: hydrolysis of tyrosine to L-DOPA, and oxidation of DOPA to DOPA quinine [2,3] . In addition, it is well-known that microphthalmia-associated transcription factor (MITF) strongly up-regulates the expression of tyrosinase and other melanogenic enzymes, such as tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2) [4][5][6] .…”

Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo

“…β-Actin was detected as loading control the suppression of MITF, which is consistent with previous findings that MITF is a transcription activator regulating those melanogenesis-related genes. [52][53][54] Furthermore, the process of melanosome transportation could be disturbed by miR-25. In that case, miR-25 is possibly involved in the entire pathological development of vitiligo, from the initial dysfunction to the final disappearance of melanocytes.…”