Everyone knows and seems to agree that melanocytes are there to generate melanin -an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin (1). What about the cell that generates melanin, then? Is this dendritic, neural crestderived cell still serving useful (or even important) functions when no-one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time -at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanoctyes matter for normal epidermal and ⁄ or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the 'secret identity' of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes (2), this critical re-examination of melanocyte biology provides a cornucopia of old, but underappreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought-provoking questions that remain to be definitively answered by future research. Praeludium pigmentosumFor those uninformed, the skin is an inert plastic wrap nature provides to keep us in and everything else out. How mistaken they are! The skin, in particular the epidermis, is one of the most active of all tissues ⁄ organs.Nature wisely placed the capillary circulation in the dermis. The epidermis has no vascular circulation thereby minimizing the probability that toxic chemicals, bacteria or fungi that penetrate through the stratum corneum can diffuse into the blood stream. That does not leave the epidermis defenseless. The epidermis has proteins called defensins that have anti-microbial properties. There are Toll-like receptors that recognize invading organisms and incite a host response. Even more interesting, it is well known that keratinocytes are avidly phagocytic. They have the capacity to phagocytize the wandering, invasive fungi or bacteria and digest them. It is both interesting and important that a-MSH stimulates the ingestion of candida by keratinocytes. a-MSH has a wide array of activities, only one of which is to stimulate the synthesis of melanin. There are receptors for a-MSH on Langerhans cells and keratinocytes as well as melanocytes. It has the ability to suppress infla...

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

Waardenburg syndrome (WS) is a hereditary disorder that causes hypopigmentation and hearing impairment. Depending on additional symptoms, WS is classified into four types: WS1, WS2, WS3 and WS4. Mutations in MITF (microphthalmia-associated transcription factor) and PAX3, encoding transcription factors, are responsible for WS2 and WS1/WS3, respectively. We have previously shown that MITF transactivates the gene for tyrosinase, a key enzyme for melanogenesis, and is critically involved in melanocyte differentiation. Absence of melanocytes affects pigmentation in the skin, hair and eyes, and hearing function in the cochlea. Therefore, hypopigmentation and hearing loss in WS2 are likely to be the results of an anomaly of melanocyte differentiation caused by MITF mutations. However, the molecular mechanism by which PAX3 mutations cause the auditory-pigmentary symptoms in WS1/WS3 remains to be explained. Here we show that PAX3, a transcription factor with a paired domain and a homeodomain, transactivates the MITF promoter. We further show that PAX3 proteins associated with WS1 in either the paired domain or the homeodomain fail to recognize and transactivate the MITF promoter. These results provide evidence that PAX3 directly regulates MITF and suggest that the failure of this regulation due to PAX3 mutations causes the auditory-pigmentary symptoms in at least some individuals with WS1.

MITF (microphthalmia-associated transcription factor) encodes a transcription factor with a basic-helix-loop-helix-zipper (bHLH-Zip) motif. MITF mutations occur in patients with Waardenburg syndrome type 2, a disorder associated with melanocyte abnormalities. Here we show that ectopic expression of MITF converts NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci of morphologically altered cells, which resemble those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase-related protein 1. Most cloned cells of MITF transfectants exhibited dendritic morphology and expressed melanogenic markers, but such properties were not observed in cells transfected with closely related TFE3 cDNA. Our findings indicate that MITF is critically involved in melanocyte differentiation.

Heme oxygenase (EC 1.14.99.3) is the rate-limiting enzyme in heme catabolism that cleaves heme at the ␣-methene bridge to form biliverdin IX␣, carbon monoxide, and iron (1, 2). Biliverdin IX␣ is immediately converted by biliverdin reductase to bilirubin IX␣ that is transported to the liver for conjugation and excretion into bile (3). There are two isozymes of heme oxygenase, heme oxygenase-1 (HO-1) 1 and heme oxygenase-2 (HO-2) (4, 5). HO-1 is inducible whereas HO-2 is constitutively expressed in human cells (6). Expression of HO-1 mRNA is highly increased in human cells by the substrate heme (7), heavy metals (8, 9), UV irradiation (10), and nitric oxide donors (11)(12)(13)(14). Because bilirubin IX␣ functions as a natural radical scavenger (15, 16), induction of HO-1 probably represents a protective response against oxidative stress. The physiological importance of HO-1 has been confirmed by the phenotypic consequences of the HO-1-deficient mice (17) and a patient with HO-1 deficiency (18).Induction of HO-1 has been extensively studied for the last few decades by many investigators. In contrast, repression of HO-1 expression has been largely ignored, despite its physiological importance (3). We have shown that HO-1 is not induced or rather reduced by heat shock in human cells (19), whereas rat HO-1 is a heat shock protein (20,21). The expression levels of HO-1 are also decreased in human glioblastoma cells by the treatment with interferon-␥ (22). In addition, hypoxia represses HO-1 mRNA expression in primary cultures of human umbilical vein endothelial cells (HUVECs), human astrocytes, and human coronary arterial endothelial cells (23). On the other hand, hypoxia increased HO-1 expression in rat liver (24) and heart (25) and in various cultured animal cells, including Chinese hamster ovary cells (26), rat ventricular smooth muscle cells (27,28), and rat myocytes (29). These results suggest the inter-species difference in the regulation of HO-1 gene expression by hypoxia between human and animal cells.The inter-species variations in the hypoxic response are of clinical significance because hypoxia is involved in the pathophysiology of various disorders, including ischemic heart disease, cerebrovascular disease, cancer, sleep apnea syndrome, and chronic obstructive pulmonary disease, which account for common causes of death and disability in the developed world. Mammalian cells respond to hypoxia in part by increased expression of several genes coding for erythropoietin (30), vascular endothelial growth factor (31), adrenomedullin (32, 33), and glycolytic enzymes (34,35), all of which cooperate to protect cells and tissues against the hypoxic state. Hypoxia-inducible

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Microphthalmia-associated transcription factor (MITF) regulates the differentiation and development of melanocytes and retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Heterozygous mutations in the MITF gene cause auditory-pigmentary syndromes. MITF consists of at least five isoforms, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, differing at their N-termini and expression patterns. Here we show a remarkable similarity between the N-terminal domain of MITF-A and cytoplasmic retinoic acid-binding proteins. To date, four isoform-specific first exons have been identified in the MITF gene: exons 1A, 1H, 1B, and 1M in the 5' to 3' direction, each of which encodes the unique N-terminus of a given isoform. The 5'-flanking regions of these isoform-specific exons are termed A, H, B, and M promoters, respectively. Among these promoters, the M promoter has received particular attention, because it is functional only in melanocyte-lineage cells and is upregulated by Wnt signaling via the functional LEF-1-binding site. Moreover, the M promoter is upregulated by other transcription factors, PAX3, SOX10, and CREB. The activity and degradation of MITF-M are regulated by extracellular signals via protein phosphorylation, such as c-Kit signaling. Together, multiple signals appear to converge on the M promoter as well as on MITF proteins, leading to the proper regulation of MITF-M in melanocytes and other MITF isoforms in many cell types.

MITF (microphthalmia-associated transcription factor) is a basic-helix-loop-helix-leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3beta selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phos-phorylation of MITF by GSK3beta and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3beta.