“Transcription physiology” of pigment formation in melanocytes: central role of MITF

Authors


Jiri Vachtenheim, Laboratory of Molecular Biology, University Hospital, Prague 8 - Bulovka, 18000, Czech Republic, Tel.: 420-266082272, Fax: 420-266082064, e-mail: jivach@upn.anet.cz

Abstract

Please cite this paper as:“Transcription physiology” of pigment formation in melanocytes: central role of (MITF). Experimental Dermatology 2010; 19: 617–627.

Abstract:  Melanin production is the primary mechanism protecting human skin against the UV light-induced damage. The polymeric compound melanin is synthesized within melanocytes in the specialized subcellular organelles, termed melanosomes, which are then transferred to surrounding keratinocytes. The genes for melanin synthesis and deposition are coordinately expressed in melanocytes. The transcription factor MITF, which has been reported to activate more than 25 genes in pigment cells, has emerged as an essential regulator not only for melanocyte development, proliferation and survival, but also for the expression of enzymes and structural proteins ensuring the production of melanin. MITF is a transcriptional activator of several genes which encode melanosome-localized proteins involved both in melanin synthesis and in melanosome biogenesis and transport, including genes whose mutations are associated with human oculocutaneous and ocular forms of albinism. Here, we outline the mechanisms of transcriptional regulation of genes associated with the biosynthesis of melanin in melanocytes and melanoma cells. MITF is crucial in this process, while several other factors seem to have only an auxiliary role to play under specific circumstances.

Abbreviations:
AIM-1

absent in melanoma

α-MSH

melanocyte-stimulating hormone α

APE-1/Ref-1

apurinic/apyrimidinic endonuclease/redox effector-1

ASP

agouti signalling protein

bHLH

basic-helix-loop-helix

Brm

Brahma

BRG-1

brahma-related protein-1

BRN2

brain-2/POU class 3 homeobox transcription factor 2

CDK

cyclin-dependent kinase

CREB

cAMP response element binding

CRF

corticotropin releasing factor

DCT

dopachrome tautomerase

DIA

diaphanous homolog 1 (DIAPH1)

EDNRB

endothelin receptor B

GPNMB

glycoprotein (transmembrane) nmb

GPR143

G protein-coupled receptor 143

HIF1α

hypoxia-inducible factor 1α

HNF-1

hepatocyte nuclear factor 1

LZ

leucine zipper

MART-1

melanoma antigen recognized by T-cells 1

MET

hepatocyte growth factor receptor

MITF

microphthalmia-associated transcription factor

ML-IAP

melanoma inhibitor of apoptosis

OA1

ocular albinism 1

OCA

oculocutaneous albinism

PKA

protein kinase A

PMEL17

melanocyte protein 17

POMC

pro-opiomelanocortin

PPAR

peroxisome proliferator activated receptor

ROCK

Rho-associated coiled-coil-containing protein kinase

RPE

retinal pigmented epithelium

Skp2

S-phase kinase-associated protein 2

SLUG

Snail homolog 2 (SNAI2)

SNF

sucrose non-fermenting

SOX10

SRY-box 10

SRY

sex determining region Y

SWI

switch

TAD

transactivation domain

TBX2

T-box 2 protein

TFEB

transcription factor EB

TFEC

transcription factor EC

TFE3

transcription factor binding to enhancer E3

TRP-1

tyrosinase-related protein 1

TRP-2

tyrosinase-related protein 2

TYR

tyrosinase

MITF as an essential transactivator in melanocytes

Pigmentation of the skin in humans and coat colour in animals are prominent phenotypic features depending on the type, mass and distribution of melanin, a polymeric compound deposited in melanosomes. In the skin, only melanocytes are specialized to produce melanin, but melanin-synthesizing cells are present also in the eye (in the retinal pigmented epithelium, choroid and iris), inner ear, leptomeninges, brain and heart. Several enzymes act sequentially to synthesize this dark polymer, which is deposited in a highly organized fashion together with the melanosomal structural proteins to form a typical dense structure of mature melanosomes.

Although the biochemical pathway from tyrosine to the melanin polymer has been known for decades (1,2), the findings of the last fifteen years have led to unveiling the transcriptional regulation of genes which are necessary for the synthesis and deposition of melanin. Remarkably, only one transcription activator emerged as a universal regulator of expression of several proteins accomplishing the assembly of the melanosome and its decoration with melanin. This protein, MITF is a product of the mi locus in mice, and numerous mutations in this locus are associated with different phenotypes affecting coat colour, hearing and eye development in mice (3–7). Mutations in this locus cause the Waardenburg syndrome type II in humans (8). MITF targets comprise not only pigmentation-associated genes. The expression of an increasing number of genes with divergent and even opposing functions in the cell relies on MITF, including cyclin-dependent kinase inhibitors p21 and p16 (WAF1) (9,10), CDK2 (11), BCL2 (12), livin (13), DIA1 (14), hypoxia-inducible factor 1α (HIF1α) (15), MET (16), T-box 2 protein (TBX2) (17), SLUG (18) and apurinic/apyrimidinic endonuclease/redox effector-1 (APE-1/Ref-1) (19) (Fig. 1a). Recently, beyond the already large group of genes activated by MITF, other potential targets were identified by using the microarrays in MITF-overexpressing human melanoma cells (20). Because severe MITF mutations preclude the development of embryonic melanocytes (21) and MITF is required for the survival of adult and even malignant melanocytes (22), the MITF protein is believed to be an essential regulator of the life and differentiation of melanocytes.

Figure 1.

 (a) microphthalmia-associated transcription factor (MITF) regulates diverse biological processes in pigment cells by activating expression of its downstream genes. Refer to text for further description. (b) Schematic view of human MITF protein. Functional domains of the molecule and the known phosphorylation sites (serines), ubiquitination and sumoylation sites (lysines) are depicted. MITF has two well-defined transcription activation domains (TAD) and a bHLH structure followed by LZ with four leucines. So far, no specific functions have been associated with the N-terminus or the C-terminal region of MITF. The full-length protein contains 419 amino acids, but a splicing variant lacking the six amino acids (ACIFPT) upstream of the basic domain was also detected.

Melanomas generally express high levels of MITF, although these levels differ greatly among melanoma cell lines and cells in the tumor tissue. MITF gene has been found to be amplified in a fraction of melanomas and is regarded as a lineage survival protein with a pro-oncogenic function in melanomas (22,23). MITF controls not only expression of pigmentation-related genes but also genes involved in diverse biological processes in melanoma cells (Fig. 1a) such as proliferation, invasiveness, resistance to apoptosis and stress mediated by reactive oxygen species, and possibly metastasis. It has been suggested that MITF protein level inversely controls invasiveness and proliferation of melanoma cells through upregulation of DIA1, the human homolog of Drosophila Diaphanous (14). High expression of MITF activates DIA1 expression, accompanied by increased proliferation via accelerated degradation of a CDK inhibitor p27 by S-phase kinase-associated protein 2 (Skp2), a DIA1-regulated gene. By contrast, low MITF level favours decreased proliferation but increased invasiveness via the Rho-ROCK-dependent pathway when DIA1-mediated actin polymerization is attenuated (14). The resistance of melanoma cells to apoptosis is at least partly mediated by expression of BCL2 and livin (ML-IAP), both of which are MITF transcriptional targets (12,13). Another important MITF target is the protooncogene MET, a protein tyrosine kinase and receptor for the hepatocyte growth factor (16), which is involved in antiapoptosis and invasion of melanoma cells (16,24). Furthermore, TBX2, a member of the T-box family of developmentally important transcription factors, plays a role in suppressing senescence and maintaining proliferation of melanoma cells (25).

The MITF-encoding locus on chromosome 3p (26) encompasses multiple promoters (at least nine), each followed by the first exon which is then spliced into a shared sequence (exons 2–9). Thus, reflecting tissue specificity, the MITF isoforms arise from different promoters which are selectively activated in melanocytes, macrophages, osteoclasts, heart muscle, or retinal pigmented epithelium (RPE) (27,28). The melanocyte-specific promoter (M-MITF) is closest to the common sequence, and the melanocyte-specific exon 1 is very short, coding only for 11 amino acid residues. The melanocyte-specific MITF isoform, MITF-M, is a 419-residue protein (Fig. 1b). MITF is a typical basic-helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor with the bHLH region followed by a leucine zipper located in the central part of the molecule while the strong transcription activation domain is at the N-terminus (N-TAD). The N-TAD was narrowed to several amino acid residues (aa. 114–132 with the core motif IISLE) which are essential for the interaction with the CH3 region of p300/CBP coactivators (29). MITF possesses also the second transactivation domain (TAD) placed at the C-terminus which is much weaker then N-TAD; nevertheless, the removal of both TADs is necessary to completely disrupt the transactivation potential of MITF. In fact, the MITF molecule in which both TADs are deleted acts in a dominant negative fashion in that it inhibits the activity of the wild-type protein (30).

Similarly, as other bHLH transcription regulators, MITF recognizes the nucleotide sequence CANNTG (E-box) but the association is maximal only if this motif is 5′-flanked with T (on either strand) (31). These T-flanked boxes were identified as essential cis-acting elements in virtually all MITF-responsive promoters. MITF is believed to bind the cis-element as a homodimer to stimulate the target genes in pigment cells. However, it is also able to form heterodimers with the related factors transcription factor binding to enhancer E3 (TFE3), transcription factor EC (TFEC) and transcription factor EB (TFEB) in vitro, and these dimers are capable of binding the E-box in gel shift assays (32). As there are no additional data to substantiate the physiological significance of these heterodimers in melanocytic transcriptional regulation, it seems unlikely that the TFE proteins play any major role in regulating the pigment formation.

At least four different transcription factors participate in the transactivation of the MITF gene in melanocytes: the paired box-containing transcription factor PAX3, a sex determining region Y (SRY) family member SOX10, the Wnt/β-catenin pathway effector LEF-1 and the cAMP pathway effector cAMP response element binding (CREB) (Fig. 2), with the last two proteins being probably more important for the maintenance of MITF levels in melanoma cells (regulation of MITF expression has been reviewed previously, e.g. in 7,27,33–35). The cut-homeodomain transcription factor Onecut-2 (36) and activated PPARγ (37) were also shown to stimulate MITF promoter. In addition, the POU domain/homeobox transcription factor hepatocyte nuclear factor 1(HNF1α) may also be involved in MITF regulation via binding to its enhancer (38), and the dimerization partner of HNF1α, DcoH (pterin-4α-carbinolamine dehydratase) was shown to be overexpressed in melanoma when compared with nevi (39). Intriguingly, DcoH is an enzyme catalysing regeneration of tetrahydrobiopterin, a cofactor of phenylalanine hydroxylase and tyrosine hydroxylase, and an allosteric inhibitor of tyrosinase (reviewed in 38). A recent report describes a direct transcriptional repression of the MITF promoter by a POU domain-containing factor brain-2/POU class 3 homeobox transcription factor 2 (BRN2) (40); however, this protein is nearly absent in normal melanocytes suggesting that its possible importance as a regulator of MITF expression is restricted to melanomas.

Figure 2.

 Regulation of microphthalmia-associated transcription factor (MITF) expression and activation of its target genes related to pigment formation in melanocytes. Arrows show transactivation, horizontal bars show transcriptional repression. Transcription is designated by stair-step horizontal arrows. Dashed arrows denote non-transcriptional regulation. Besides pigment-related genes shown here, additional MITF targets regulating cell proliferation, apoptosis, or invasiveness of melanoma cells were identified (see Fig. 1). In addition to melanocortin one receptor (MC1R), at least two other receptors, β2-AR (β2 adrenergic receptor) and MC4R (melanocortin 4 receptor), have been reported to be involved in cAMP signalling (132,133), but it is unknown whether their expression is regulated by MITF.

In addition to the transcriptional control, the MITF level is regulated by protein degradation. Clearly, phosphorylation of two serine residues (S73 by ERK2 and S409 by RSK) after the stimulation of cells with the c-kit ligand Steel leads to protein destabilization (41), presumably via the enhanced ubiquitination on lysine 201 followed by degradation by a proteasome (42). The phosphorylation at S73 was shown to be necessary for the interaction with the p300 coactivator (43). However, the truncated MITF construct lacking the N-terminus including S73 or the S73A mutant lost none of the transactivation potential (44), indicating that interaction of MITF with p300 is not a critical event for constitutive activation. More likely, it might play a physiological role in transient signal-induced changes of MITF activity. Other posttranslational modifications include phosphorylation on S298 (45), an activating event, and sumoylation on K182 and K316 (46), which may affect the activity differently depending on the target promoter (47). Nonetheless, the current data do not point to a specific covalent modification on the MITF molecule such as would critically activate or disable its function. What they do suggest is that adjustment of the intracellular level of the protein, the degradation of which is controlled by phosphorylation and ubiquitination, may be a decisive mechanism regulating the pigment production and other MITF-modulated processes such as proliferation or cell survival (as mentioned above) in normal and malignant melanocytes.

Regulation of transcription of the three melanogenesis-controlling enzymes

Tyrosinase (TYR), the first rate-limiting and tri-functional enzyme in the melanogenic biochemical route, has been studied as a prototypic target among the MITF-regulated genes. TYR catalyses the conversion of tyrosine to dopaquinone and the oxidation of DOPA (formed at the redox exchange between dopaquinone and cyclodopa, a cyclization product of dopaquinone) back to dopaquinone (48,49). Thus, DOPA acts as a cofactor which greatly accelerates the first tyrosinase-catalysed step. Dopaquinone is also the branching point between eumelanogenesis and pheomelanogenesis; it reacts with cysteine to form cysteinyldopa, a precursor of red pheomelanins. A second enzyme in the pathway, tyrosinase-related protein 2 (TRP-2) (DCT, dopachrome tautomerase), enables a rapid conversion of dopachrome (50), a cyclodopa-derived quinone, to 5,6-dihydroxyindol-2-carboxylic acid (DHICA). The last enzyme, tyrosinase-related protein 1 (TRP-1), is a DHICA oxidase (51) which facilitates the formation of carboxy group-containing eumelanins. However, in the absence of TRP-2, dopachrome is decarboxylated to dihydroxyindol (DHI) which is also polymerized to melanin (the DHI-melanin is darker than DHICA-containing melanin). DHI can serve as a third substrate for tyrosinase (52), which thus promotes also this last oxidative reaction in the pathway. Melanogenesis is spatially restricted only to melanosomes because it is a potentially toxic pathway producing several highly reactive intermediates (quinones). These can react with and damage the critical cellular macromolecules if synthesized in an inappropriate place in the cell (53–55).

All of the three enzymes operating in melanogenesis have been shown to be transcriptional targets of MITF (Fig. 2) with an identical TCATGTG sequence in their proximal promoters quite close to the start of transcription. Promoter-reporter studies revealed that promoters of tyrosinase (56,57), TRP-1 (58,59) and TRP-2 (58) were activated by cotransfected MITF. The human tyrosinase promoter contains the M-box (extended E-box, AGTCATGTGCT) located about 100 bp upstream of the transcription start and the E-box sequence ACATGTGA at the initiator. Paradoxically, not the M-box but this initiator E-box is more important for the promoter function (56). The identified well-defined MITF-binding motifs in the tyrosinase promoter and in other promoters of MITF-responsive genes are summarized in Table 1.

Table 1.   Functionally relevant MITF-binding motifs in proximal promoters of MITF-regulated human genes which participate in the production and deposition of melanin
Target geneM-box or E-boxPosition relative to the transcription startReference
  1. MART-1, melanoma antigen recognized by T-cells 1; OA1, ocular albinism 1.

  2. 1Human genome searches: http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606).

TYRAGTCATGTGCT−107 to −9756
ACATGTGA−13 to −6
TRP-1AATCATGTGCT−211 to −20166
TRP-2GGTCATGTGCT−137 to −12766,75
GPNMBGCACATGAGT−46 to −37101, (http://www.ncbi.nlm.nih.gov/Genomes)1
Melastatin 1GCTCACATGCT−63 to −53112 (http://www.ncbi.nlm.nih.gov/Genomes)
MART-1TCACGTGTG−715 to −70799, 130 (http://www.ncbi.nlm.nih.gov/Genomes)
PMEL17TCACATGAA+ 583 to + 59199 (http://www.ncbi.nlm.nih.gov/Genomes)
OA1GGCACATGATG−33105
RAB27AACAGCTGA−46 to −39117,131
CCATATGA−58 to −51

While MITF ensures a continuous transcription of the tyrosinase gene under normal circumstances and after hormonal stimulation through the cAMP signalling, the increase of tyrosinase production as the UV response is mediated, at least in part, by another bHLH factor, the ubiquitous USF-1 (60). Upon UV irradiation, USF-1 is phosphorylated by the p38 stress kinase and this modification is required for the USF-1-mediated transactivation of the tyrosinase promoter (60). In addition, the p53 tumor suppressor protein was also shown to participate in increased melanogenesis after UV irradiation because p53 upregulates the tyrosinase and TRP-1 promoters in reporter assays, and potential binding sites for p53 were identified in the TRP-1 promoter (61). Further, tyrosinase mRNA level was increased via a p53-dependent mechanism upon UV irradiation of melanoma cells in culture, and p53 was required for the thymidine dinucleotide-induced increase of tyrosinase in mouse epidermis (62). Another transcription factor, a dimer of DcoH/HNF-1alpha, was also found to be involved in tyrosinase transcription in skin melanocytes (63). According to Hou and coworkers, the mouse embryonic melanocytes require a coordinated action of Mitf and Sox10 for tyrosinase induction, because both pigmentation and tyrosinase expression in Sox10-deficient neural tube explant cultures were rescued only by exogenous Sox10, which acts upstream of MITF, but not by exogenous MITF alone (64). The MITF-related bHLH protein TFE3, the closest to MITF in evolution, was reported to bind E-boxes of both TYR and TRP-1 promoters in vitro. Also, exogenous TFE3 up-regulated the promoter-reporters (65). However, because endogenous TFE3 failed to form dimers with MITF or bind to the M-boxes, its physiological role in melanogenesis is disputable.

The promoter of the human TRP-1 gene possesses the M-box (AATCATGTGCT) which is localized about 210 bp upstream of the start of transcription and, unlike the mouse promoter, the human one harbours the TATA sequence (66). While the M-box is necessary for promoter upregulation by MITF (58), the TRP-1 promoter is contacted and its activity is elevated also by Pax3 (67), the MITF promoter-activating transcription factor in melanocytes. Furthermore, TBX2, a T-box protein family member expressed in pigmented cells, is capable of transrepressing the TRP-1 promoter (68).

In what is an intriguing feature of the TRP-1 gene, its transcription is frequently and selectively attenuated or completely extinguished in melanoma cell lines and tumor tissues, whereas tyrosinase, TRP-2 and MITF itself are normally expressed. Weaker expression of TRP-1, as determined by in situ hybridization, was also revealed in nevi: the lower dermal layer of intradermal nevi showed a reduced signal when compared with the expression in the upper dermal layer, while a high expression was detected in the basal epidermal layer in junctional nevi (69). Similarly, in dermis-invading lesions of primary melanoma, the staining with the anti-TRP-1 antibody was also fading (70). Transcriptional blockage was observed upon the addition of a known inducer of differentiation hexamethylene bisacetamide (HMBA) to the medium of melanoma cells in culture (71). A complete extinction of TRP-1 was seen in several human melanoma cell lines (72–74). Although lower MITF binding to the TRP-1 E-box was observed in TRP-1-negative cell lines (73), this cannot account for the complete loss of TRP-1 mRNA. Thus, the mechanism which may involve a putative TRP-1-specific repressor remains to be elucidated. This might be of importance because TRP-1 is a melanocytic antigen recognized by activated T-lymphocytes; consequently, TRP-1-negative melanomas can evade eradication by DNA vaccines directed to TRP-1 epitopes.

TRP-2 enzyme (DCT) is expressed very early during melanoblast differentiation in the developing embryo, approximately when MITF begins to be expressed. The 5′-regulatory region of the TRP-2 gene contains the M-box (GGTCATGTGCT) positioned about 135 nucleotides upstream of the transcription start (75) and the promoter responds to MITF coexpression (58). Another important factor specifically involved in the transcription of TRP-2 is SOX10, which also promotes expression of MITF (see above). The human TRP-2 promoter-reporter construct was shown to be activated by SOX10 (76,77) and SOX10 and MITF (Fig. 2) act in a synergistic manner to activate the promoter (78,79). These data are in accord with the observation that mouse heterozygous embryos carrying the Sox10dom mutation transiently lack Dct (around days 11–12) in the melanoblast lineage, and MITF alone is incapable of triggering the Dct transcription in these early MITF-positive cells (77). In a recent report, another member of the Sox family, Sox5, inhibited the Sox10-stimulated activity of the Dct promoter in melanocytes (80). In the TRP-2 expression, yet another transcription factor, LEF-1, is thought to play a role in conjunction with MITF. LEF-1 protein, one of the effectors of the Wnt/β-catenin pathway at target promoters, was shown to physically interact and cooperate with MITF in the transactivation of TRP-2 promoter. However, not only the MITF-LEF-1 interaction, but also the cis-acting motif in the promoter (surprisingly, not recognized by LEF-1) are required for the TRP-2 promoter stimulation (81). A CRE-like element in the TRP-2 promoter might also contribute to gene expression through direct regulation by CREB protein (58).

The distal enhancers were described for mouse tyrosinase and Trp1 genes (82). An upstream regulatory region for human tyrosinase, too, is reportedly located about −9 kb upstream from the start of transcription (83,84). This sequence, which showed a homology with a similar distal locus found in the mouse and functioned as an enhancer in transfection assays, may prove to be important for the pigment cell-specific expression of human tyrosinase.

It is also noteworthy that the three human promoters of melanogenic enzymes contain either a typical TATA box (tyrosinase and TRP-1 promoters) (66,85) or a TATA-like element (TRP-2 promoter) (66,75), but their function in tissue-specific transcription is presumably omissible. At least in the case of tyrosinase, the TATA sequence seems to be superfluous for the promoter activity in reporter assays (our unpublished results). Although other recognition elements are also present in the tyrosinase, TRP-1 and TRP-2 promoters, none have been shown to be crucial for expression and the promoter deletion analyses pointed to the MITF-binding elements as regulators of the promoters activity with MITF being the essential physiological transactivator.

MITF conveys hormonal and stress responses to pigmentation genes

The pigmentation is stimulated hormonally by α-MSH and its receptor melanocortin one receptor (MC1R) via the cAMP signalling cascade. The POMC (pro-opiomelanocortin) gene encodes a precursor that is processed to form the α-MSH, ACTH and β-endorphin. The POMC gene is active mainly in the pituitary, but POMC-derived peptides are produced also in peripheral tissues including skin melanocytes (35,86). Although α-MSH is a potent inductor of pigmentation in mammalian skin, β-MSH and ACTH possess melanogenic activities as well (hormonal regulation of skin pigmentation has been reviewed, e.g. in 35,87,88).

The hormonal stimulation by α-MSH is mediated through the MC1R resulting in increased intracellular cAMP level and activation of protein kinase A (PKA). This notorious hormonal signalling pathway has specific consequences in melanocytes as the MITF promoter possesses a CRE element which is bound by CREB proteins that are phosphorylated by PKA and activate transcription of MITF. It is not surprising, therefore, that pharmacological agents that increase the cAMP level in the melanocyte are extremely potent in upregulating the endogenous level of MITF, which consequently triggers transcription of downstream melanogenic factors. Although increase of MITF mRNA and protein is only a transient response, the resulting induction of melanogenesis is a prominent outcome of this hormonally triggered signalling cascade. It is noteworthy that MSH can control melanogenesis also independently of MC1R, possibly by acting directly in melanosomes (38). Remarkably, the MC1R gene was identified as an MITF target (Fig. 2), as its promoter-reporter activity responded to MITF cotransfection in human melanoma cells (89). Thus, a positive feedback loop is created after hormonal stimulation resulting in an increased expression of receptor molecules in the pigment cell. Intriguingly, yet another receptor is upregulated by MITF; the expression of endothelin receptor B (EDNRB), which serves as a receptor for peptides endothelins, is also regulated by MITF (90). Moreover, signalling by endothelins 1 and 3 activates MAP kinases with subsequent phosphorylation of MITF and stimulation of MITF expression as well (90). As with the α-MSH signalling, endothelins induce CREB protein phosphorylation with consequent activation of MITF expression in this pathway.

Hormonal signalling is also involved in skin responses to UV irradiation, although the effect of UV light on pigmentation is complex. UVB can induce expression of POMC and MC1R genes in melanocytes and keratinocytes (87,91). In addition, expression of corticotropin-releasing hormone (CRH) is also stimulated by UVB in melanocytes, which is mediated by the CREB-PKA signalling with consequent stimulation of POMC expression through the CRH-R1 receptor (91). The POMC gene has been reported to be p53-responsive following UV irradiation, and the POMC promoter contains a p53 binding site which is necessary for its highest activity (92). The in vivo tanning response and POMC mRNA induction were dependent on p53, further suggesting that the p53 protein is an important mediator of UV-induced melanogenesis increasing the transcription of POMC (92). However, the concept that activated p53 is the only essential stimulator of UV-induced pigmentation via induction of POMC transcription was challenged from several reasons, mainly because the POMC-knockout mice have been known to display normal melanin pigmentation, and a more complex regulation was suggested (93). A direct regulation of tyrosinase transcription after UV irradiation is mentioned above.

Hormonal regulation also underlies the switch of pigment type formation in skin. The α-MSH signalling on the MC1R receptor is inhibited by the agouti signalling protein (ASP, ASIP in humans) that can compete with α-MSH in binding to MC1R. High expression of ASP is associated with yellow bands in mouse hair. Thus, MC1R and its ligands, α-MSH and ASIP, regulate switching between eumelanin and pheomelanin synthesis in melanocytes (35,94). ASP was demonstrated to downregulate MITF gene expression, and hence its targets, by antagonizing the effect of α-MSH, thus favouring pheomelanogenesis by reduced production of eumelanin (95). Interestingly, a similar profile of genes was inversely regulated by ASP and α-MSH in a microarray analysis (96). Recently, it has been shown that the receptor-binding domain of ASIP efficiently antagonizes the MSH-MC1R signalling by reducing the cAMP level, while it induces no changes in pigmentation, demonstrating that the negative regulation of differentiation by agouti signalling is independent of the cAMP-CREB pathway (94). Other hormones such as steroids can also influence pigmentation (reviewed in 35,87), and a recent report describes that even cholesterol is capable of increasing expression of MITF and its target genes in melanocytes, possibly through the upregulation of the CREB protein (97).

MITF regulates expression of non-enzymatic melanosomal proteins

Biogenesis of melanosomes

Early stage melanosomes (stage I and II) display a striatal structure formed by amyloid-like fibrils upon which the polymer melanin is deposited in later stages (stage III and IV). PMEL17 (also known as SILV or GP100), a critical structural protein required for the formation of the characteristic fibrilar structure, can by itself drive the formation of fibrilar structures even if expressed exogenously in non-pigmented cells (98). Pmel17 is a product of the mouse silver locus, and the human SILV protein is recognized by the widely used melanoma diagnostic antibody HMB-45. In melanoma cells, PMEL17 was found to be a MITF target (Fig. 2) with protein and mRNA levels correlating well with those for MITF, and PMEL17 mRNA was upregulated upon transfer of wt MITF and downregulated by a dominant-negative MITF mutant (99). The MITF-binding site critical for the promoter activity is located, quite exceptionally, in the first intron and conforms the TYR family consensus TCATGTG. The Pmel17 transcript was not detectable in RPE of MITF-mutant mouse embryos, further supporting the necessity of MITF expression for the Pmel17 gene transcription (100).

Recently, a transmembrane glycoprotein osteoactivin (GPNMB), expressed in osteoclasts, macrophages and melanoma cells, has been identified as a MITF-regulated gene both in pigmented cells (101) and in osteoclasts (102). Although Gpnmb manifests partial similarity to the silver protein and localizes to melanosomes, its precise role in melanocyte physiology is not known. Mouse Mitf mutant embryos do not express Gpnmb while in normal embryos, the protein appears early in the development of RPE and melanoblasts. The E-box CACATGA in the Gpnmb promoter is located next to the AP1 site and is highly conserved during evolution. This motif is important for the reporter expression in transfected cells; however, even the E-box-deleted construct was capable of directing the transgene expression in zebrafish melanoblasts, indicating that the remaining sequences in the short (89 bp) promoter are sufficient for in vivo expression (101).

MLANA (also termed melanoma antigen recognized by T-cells 1(MART-1) or Melan-A) is yet another transmembrane protein associated with melanosome biogenesis. MLANA is in a complex with PMEL17 and influences its stability and processing within the melanosome (103). MLANA has also been recognized as a MITF transcriptional target (Fig. 2) with two E-boxes in the human promoter, the more proximal one being the TCACGTG motif (99). MLANA promoter activity depends on cotransfected MITF and is bound by MITF in chromatin immunoprecipitation assays. The MLANA protein and mRNA levels correlate with MITF levels in melanoma cell lines (99). Thus, the expression of this melanosomal matrix protein is also believed to require MITF.

The ocular, X-linked albinism type 1 is caused by mutations in the ocular albinism 1(OA1) gene (also named GPR143) (104). Its protein product is a melanosome membrane protein expressed exclusively in melanocytes and RPE cells. OA1 shares a similarity with members of a superfamily of the G protein-coupled receptors. Melanocytes carrying the mutated OA1 gene display giant abnormal melanosomes, implicating its product in melanosome biogenesis. One evolutionarily conserved binding site for MITF (CACATGA), only 28 nucleotides upstream from the transcription start, was found in the mouse Oa1 gene promoter (nt -33 in the human promoter) (105). Thus, as the target sequence on the opposite strand is in keeping with the classical consensus motif TCATGTG, the mouse Oa1 promoter (and very likely also the human OA1 promoter) was assumed to be a direct target regulated by MITF in pigment cells (Fig. 2). The integrity of this site was indeed essential for the promoter activity and binding to MITF in gel shift assays (105). MITF protein also occupied the human promoter in melanoma cells. As with several other MITF target genes, a relatively short fragment of the proximal Oa1 promoter (containing the E-box) was sufficient to direct the tissue-specific expression: when introduced into mouse retina, the transgene driven by this promoter sequence was expressed in RPE (105).

Another transporter protein localized in melanosomes is AIM-1 (absent in melanoma, also called MATP for membrane-associated transporter protein, and also designated as SLC45A2). Mutations of this gene were shown to cause OCA4 (106). MATP protein like another melanosomal protein, the P protein regulating the intramelanosomal pH (107), contains 12 transmembrane regions and is required for a correct tyrosinase processing in melanosomes, because homozygously mutated mouse melanocytes (uw/uw) display a defect during maturation of melanosomes, which are only slightly melanized, irregularly shaped and have a disorganized fibrilar structure (108). Remarkably, tyrosinase (and also TRP-1 and TRP-2) are secreted from uw/uw mutated melanocytes in vesicular bodies. Human AIM-1 gene contains a repetitive sequence in the 5′-regulatory region with numerous E-boxes, including the consensus TCATGTG sequences, and ectopic MITF upregulates AIM-1 mRNA in human melanoma cells (109). Nevertheless, the positive regulation of Aim-1 might be indirect as the endogenous MITF does not seem to associate with the Aim-1 promoter (109).

Two members of a large family of transient receptor potential (TRP) proteins, melastatin 1 (TRPM1) and melastatin 7 (TRPM7), were found expressed in melanomas and nevi. While melastatin 1 (also known as MLSN1) was expressed in normal melanocytes, nevi and primary melanomas, it was not detected in metastatic lesions (110). Contrary to this, TRPM7 expression was found in several metastatic melanoma cell lines (111). Whilst it is unknown whether TRP7 expression is MITF-dependent, melastatin 1 was identified as a MITF target gene, the promoter of which responded exceptionally sharply to MITF cotransfection (112,113). The human promoter region contains several E-boxes. The most proximal of these (at around nt -58) is presumably the main element important for promoter activity (112). Chromatin immunoprecipitation revealed that MITF resides on the TRPM1 promoter in melanoma cells. Furthermore, mRNA levels of TRPM1 and MITF correlated very closely both in melanoma cells and in normal melanocytes. Also, melastatin 1 mRNA was lacking in RPE derived from MITF mutant embryos, thus indicating a dependency on MITF in developing pigmented cells (112).

Melanosome transport

Movement towards the cell periphery and transfer to the surrounding keratinocytes mark the physiological route of melanosomes in skin melanocytes. Molecular interaction essential for the melanosome trafficking in the dendrite tips results in the formation of trimolecular complex consisting of RAB27A, a small GTPase protein, its effector melanophilin (SLAC2A), and an actin-based protein myosin Va (114–116). MITF was shown to upregulate the transcription of the RAB27A gene and its promoter-reporter and, conversely, siRNA-mediated knockdown of MITF was found to impair the normal distribution of melanosomes in mouse B16 melanoma cells via downregulation of Rab27a (117). The MITF-dependent transcriptional regulation of RAB27A (Fig. 2) is phylogenetically conserved because MITF was also required for melanosome transport and dendricity in Xenopus melanophores (118). Notably, the expression of melanophilin, myosin Va, as well as another GTPase Rab7, which is involved in the microtubule-based transport of early-stage melanosomes (114) and in the maturation of TRP-1 and Pmel17/Silv proteins within the melanosome (119,120), was found not to be influenced by MITF (117).

OA1 gene transcription gene is directly activated by MITF (see above). Besides its function in the biogenesis of melanosomes, studies on mouse melanocytes demonstrated the Oa1 protein′s importance for melanosome transport to the cell periphery (121). MITF therefore regulates melanosome traffic by sustained expression of at least two proteins, Rab27a and Oa1.

Additional specialized proteins have been identified in the Golgi-lysosome-melanosome network. These include RAB38 (chocolate in mice), the P protein (its defects were found in OCA2, known as pink-eyed in mice), melanophilin, SLC24A5 (potassium-dependent sodium-calcium exchanger), and several proteins of the BLOC (biogenesis of lysosome-related organelles complexes), which are altered in Hermansky–Pudlak syndrome subtypes (122). In spite of that, the mechanism of their transcription and possible MITF involvement still await elucidation.

MITF cofactors and regulation of endogenous genes

The melanosomal enzymes and non-enzymatic proteins, the transcription of which is controlled by MITF, constitute only a subset of MITF-responsive genes. It is less clear whether MITF utilizes the same cofactors, coregulators, or cooperates with additional transcription factors to specifically regulate so many target genes involved in so diverse cellular processes in pigment cells. The p300 and CBP proteins, which are global transcription coactivators serving as cofactors for many different transcription factors (123), were found to bind MITF and stimulate transcription from the responsive promoter-reporters (29,43). Therefore, they are presently thought to be MITF coactivators. P300/CBP proteins are histone acetyltransferases capable of acetylating histones and non-histone proteins, many of which are transcription factors. Acetylation of N-terminal lysine residues on histone tails by p300/CBP and other histone acetyltransferases neutralizes the negative charge and reduces the interaction between histones and DNA. Although it is likely that MITF recruits p300/CBP to target promoters causing the hyperacetylation of surrounding histones, MITF itself might be acetylated by these, and possibly by other, histone acetyltransferases. MITF has several potential acetylation sites and is acetylated in vitro (our unpublished results), but whether the acetylation influences its activity or helps to discriminate among the many targets remains to be established.

Endogenous genes often require local chromatin decondensation in the promoter regions. This is accomplished, besides the histone acetylation, by the action of chromatin remodelling complexes. The SWI/SNF complex which utilizes Brm or Brg1 ATPases is necessary for the efficient transcription of endogenous Tyr, Trp-1, dct and Pmel17 genes in mouse fibroblasts transfected with MITF, because expression of these genes was found attenuated by dominant negative mutants of Brm and Brg1 (124). On the contrary, the MC1R gene expression was not decreased by these mutants. Thus, the expression of differentiation-specific genes might require the SWI/SNF enzymes in melanocytes. Transcription of TYR, but not other markers, can be triggered by MITF also in the human non-melanocytic (osteosarcoma) cell line U2OS (74), but not in H1299 lung carcinoma cells, in which Brg1 is not expressed. However, co-delivery of exogenous Brg1 together with MITF into H1299 cells elicits the expression of endogenous tyrosinase (125), indicating that a similar requirement of SWI/SNF for the tyrosinase expression exists in human cells.

As MITF is a crucial activator of melanogenesis-related genes, an increase or decrease of its level should be reflected by changes in transcriptional activity of the downstream genes. While it is believed that the inhibition of MITF expression or activity leads to the decreased levels of its targets and eventually to the proliferation block or induction of apoptosis in melanoma cells (12,22), exogenously increased MITF level does not have any effect on tyrosinase or Trp-1 expression (126). So, perhaps, additional factors or coactivators may be required as well as MITF, or some unknown mechanism may restrict the production of pigment cell markers at constant lower levels and only stress conditions such as UV light might trigger the specific coordinated response to facilitate melanogenesis.

Interaction with other proteins, as demonstrated for β-catenin, could be another level of regulation of MITF activity on the downstream promoters (127). Transcription of MITF itself is up-regulated by the Wnt/β-catenin pathway, which is frequently deregulated in many cancers including melanomas. The identified direct interaction between MITF and β-catenin can result in the redistribution of β-catenin from LEF-1 target promoters to MITF targets. MITF can thus exploit β-catenin as its own cofactor in melanomas (127). Last, the Rb tumor suppressor protein was reported to interact with MITF (128) and activate transcription of the MITF target p21(WAF1), thus causing cell cycle arrest (9). Although the Rb−/− mouse melanocytes displayed pigmentation defects in vitro (129), it remains unclear whether Rb directly participates in the transactivation of melanogenic factors.

Conclusions

The published data lead to a notion that MITF represents a melanocyte-specific communication hub integrating intracellular signals and transcription responses in cell differentiation, proliferation and survival. The synthesis of melanosomal enzymes and structural proteins is coordinated already at the transcriptional level because MITF regulates their gene promoters. Hormonal and UV light-induced changes in pigmentation seem to be more complex and are further regulated by transcription factors p53 and USF-1. In addition to pigmentation, MITF regulates also proliferation, survival and invasiveness of malignant melanocytes by regulating transcription of genes involved in these processes. Further experimentation is needed to elucidate how this central position of MITF in the melanocyte physiology influences the sets of the transcriptional targets to be regulated according to the requirements of the pigment cell.

Acknowledgements

This work was supported by grant NR/9319-03 (Ministry of Health) and from institutional projects MZO00064211 (MH) and MSM21620808 (MEYS), Czech Rep.

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