Microphthalmia-associated transcription factor modulates expression of NADPH oxidase type 4: A negative regulator of melanogenesis

Abstract

How signaling via reactive oxygen species (ROS) influences skin pigmentation is unclear. We have investigated how NADPH oxidase-derived ROS modulates the expression of the key pigment “melanin” synthesizing enzymes in B16 mouse melanoma cells. A melanin inducer α-melanocyte-stimulating hormone (α-MSH) caused ROS generation that was inhibited by the NADPH oxidase inhibitor Diphenyleneiodonium (DPI) and was insensitive to antagonists of other ROS-producing enzyme systems including mitochondrial enzymes, cycloxygenase, and xanthine oxidase. NADPH oxidase 4 (Nox4) was found to be the most abundant isoform expressed in B16 cells, and its gene levels, as well as ROS generation, were enhanced by α-MSH. Interestingly, silencing Nox4 gene expression with Nox4 siRNA augmented melanin formation under basal conditions and after α-MSH stimulation, demonstrating that constitutive or stimulated Nox4-dependent ROS inhibits melanin formation. This process may be mediated by targeting the promoter region of a melanin synthesizing enzyme tyrosinase, because Nox4 siRNA enhanced tyrosinase promoter activity. Moreover, inhibition of tyrosinase mRNA expression in Nox4 siRNA-treated cells by blocking de novo mRNA and protein synthesis with actinomycin D and cycloheximide respectively indicates that Nox4 repression induces melanogenesis by increasing tyrosinase gene expression. We also found that α-MSH activated its downstream signal transducer microphthalmia-associated transcription factor (MITF) to stimulate Nox4 gene expression. We thus identified a novel mechanism by MITF signaling that in turn stimulates Nox4 to drive ROS generation, thereby repressing melanin synthesis. Such sequence of actions appears to act as an internal feedback mechanism to fine-tune melanin synthesis in response to exogenous challenges such as UV radiation.

Introduction

Skin pigmentation is the major photoprotective mechanism against the carcinogenic and aging effects of solar irradiation. Epidermal melanocytes synthesize the pigment melanin, in the form of eumelanin or pheomelanin. Synthesis of the photoprotective eumelanin by human melanocytes is regulated mainly by the alpha-melanocyte stimulating hormones (α-MSH) which bind to the melanocortin 1 receptor (MC-1R) and activate the cAMP pathway that is required for UV-induced tanning [1]. In mammalian melanocytes, melanins are synthesized within melanosomes that contain three major pigment synthesizing enzymes: tyrosinase, tyrosinase-related protein-1 (TRP-1), and dopachrome tautomerase (DCT), also known as tyrosinase-related protein-2 [2], [3], [4].

Molecular and genetic data suggest that the activation of MC-1R plays a crucial role in pigmentation in humans and mice [5], [6]. α-MSH cleaves from pro-opiomelanocortin (POMC), a multicomponent precursor for α-MSH (melanotrophic), ACTH (adrenocorticotropic hormone; adrenocorticotrophic), and the opioid peptide β-endorphin [7], by binding to MC-1R, and up-regulating cAMP production leads to melanocyte differentiation. This is with respect to upregulated cellular activities such as melanin synthesis [8], dendrite outgrowth [9], and melanosome transportation [10], some of the major events contributing to pigmentation. In all of these regulatory pathways it has been shown that cAMP production triggers its downstream effector molecules protein kinase A (PKA) and cAMP-responsive element binding (CREB) protein 1 transcription factors, to up-regulate the expression of microphthalmia-associated transcription factor (MITF) [11], the master regulator of melanogenesis that, in turn, controls the production of melanogenic enzymes (tyrosinase, TRP-1, and DCT) [12] and melanocyte development [13]. Therefore α-MSH/MC-1R/MITF appears to be an important signaling pathway in melanogenesis.

The skin is susceptible to oxidative damage [14] and reactive oxygen species (ROS) have been implicated in melanogenesis [15]. However, ROS production has been shown to suppress pigmentation in melanoma cells and its accumulation in the epidermis is associated with vitiligo, the polygenic pigment skin disorder. Therefore the role of ROS in pigmentation remains controversial. Although excessive ROS generation may have a deleterious effect, physiological levels of ROS have an intracellular signaling role in cell proliferation, migration, apoptosis, and differentiation [16], [17]. The major sources of intracellular ROS production include mitochondria [18] and various metabolic and detoxifying enzymes such as cytochrome c oxidase [19], xanthine oxidase [20], nitric oxide synthase [21], and NADPH oxidase [22]. Of all the ROS generating enzymes, the NADPH oxidase family is the sole enzyme system whose primary biological function is to produce ROS [22]. Seven isoforms (Nox1–5 and Duox1–2) have been identified and they are classified by reference to the prototype phagocytic gp91^phox (Nox2) [22], [23], [24], [25]. Using the well-established B16 mouse melanoma model, Nox4 was found to be the most abundant isoform and its protein and mRNA levels and ROS production were all augmented by α-MSH. Interestingly, basal or stimulated melanin synthesis was potentiated by silencing the gene expression of Nox4, suggesting that ROS production is inversely correlated with melanin synthesis. Here we propose a novel negative regulatory mechanism of pigmentation in B16 cells via MITF/Nox4-derived ROS generation. This is mediated by a transient reduction of the melanin synthesizing enzyme tyrosinase. Such response may provide an internal feedback mechanism that modulates melanin synthesis in the melanogenic pathway.