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MITF
Critical Regulator of the Melanocyte Lineage
Erez Feige, Laura L. Poling, and David E. Fisher
C
ONTENTSINTRODUCTION
THE MiT FAMILY
MITF ISOFORMS
MITFAND WAARDENBURG SYNDROME
TRANSCRIPTIONAL REGULATION OF M-MITF
CRITICAL ROLES OF MITF IN THE MELANOCYTE LINEAGE
POSTTRANSLATIONAL MODIFICATIONS OF MITF PROTEIN–PROTEIN INTERACTIONS OF MITF MITFIN CANCER
CONCLUSIONS AND PERSPECTIVES
REFERENCES
Summary
The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix–leucine zipper (bHLH-LZ) protein that acts as either a homo- or heterodimer with the related proteins TFEB, TFEC, and TFE3. Several isoforms of MITF have been identified, although only M-MITF is restricted to the mel- anocyte lineage. Although MITF is important in the development of multiple cell lineages, it plays a particularly important role in melanocyte differentiation and function. MITF mediates the pigmentation process through transcriptional regulation of melanogenic enzymes and processing factors. MITF is directly involved in regulation of multiple targets, some of which are established melanoma histopatho- logical markers, as is MITF itself. As a point at which upstream signaling pathways converge, and as an activator of critical downstream cellular processes, MITF is a pivotal transcriptional regulator in the melanocyte lineage. This chapter will discuss the transcriptional and posttranslational control of MITF, will summarize the current knowledge of its target genes, and will portray our understanding of its roles in melanocytes and melanoma.
Key Words: Microphthalmia; melanocyte; melanoma; pigmentation; transcription.
INTRODUCTION
Microphthalmia-associated transcription factor (MITF) is a developmentally regu- lated and evolutionarily conserved transcription factor, with homologs identified from
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From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ
arthropods to mammals, including Drosophila (1), Xenopus (2), zebrafish (3), chicken (4), mouse (5), and human (6). Mutations in the mouse Mitf locus have demonstrated that Mitf is implicated in regulation of several cell lineages. It is essential for neural crest- derived melanocytes (5,7–9), as highlighted by the loss of melanocytes in mice (reviewed in ref.10) or melanophores in zebrafish null for Mitf (3). In addition, Mitf is important for normal development of the retinal pigment epithelium (RPE), osteoclasts, and mast cells (5,11,12), and some Mitf alleles result in small eyes, osteopetrosis, and mast cell loss. In humans, mutations in MITF are the cause of Waardenburg Syndrome type 2a (WS2a), characterized by hypopigmentation and sensorineural deafness resulting from loss of melanocytes in the skin, hair, and inner ear (13).
Several isoforms of MITF have been identified, exhibiting diverse patterns of expres- sion ranging from ubiquitous to cell type-specific, such as the restricted transcription of M-MITF in the melanocyte lineage. MITF expression, activity, and stability are tightly regulated through different promoters, alternative splicing, and posttranslational modi- fications. Data accumulating over the last decade have shed some light on the molecular mechanisms underlying MITF regulation and their roles in melanocytes. In addition, certain data suggest that MITF, or members of the MITF/TFEB/TFEC/TFE3 (MiT) family of transcription factors, are implicated in human malignancy.
THE MiT FAMILY
MITF is a member of the MiT family of basic helix-loop-helix–leucine zipper (bHLH- LZ) transcription factors (5). MITF maps to mouse chromosome 6 D3 (11) and chromo- some 3p12.3-14.1 in humans (6,13). Additional members of the MiT family are TFE3, TFEB, and TFEC, which have a similar genomic organization with conserved intron/
exon boundaries (14). The MiT members use the highly conserved basic and HLH-LZ regions to bind target DNA containing an E-box motif, CAC(G/A)TG (15), and for homodimerization or heterodimerization (15–17), respectively. The formation of heterodimers, such as MITF-TFE3 and MITF-TFEC, has been observed (15,18–20);
however, heterodimeric interactions may not be necessary for function, as suggested by redundant roles of MITF and TFE3 in osteoclast development (21). Similar to MITF, the related family members TFEB and TFEC are found as alternative splice forms with differential first exon usage, indicating possible alternative promoters (22). The relative expression levels of the MiT family members, combined with the alternative splice forms (22) and the formation of different homo/heterodimers, may contribute to tissue- specific distribution of MiT complexes.
MITF ISOFORMS
Several isoforms of MITF were identified (designated A, B, C, D, E, H, M, and Mc), which differ in their N-termini because of alternative promoters and first exons. How- ever, all of the identified isoforms share downstream exons 2–9, which include the transactivation domain, basic region, HLH-LZ, and the known posttranslational modi- fication target amino acids (13,23–26). It is therefore unclear whether the various isoforms specify distinct functional activities for MITF or whether their different 5' ends only specify tissue expression patterns. Multiple cell types express MITF; A-, B-, C-, and H-MITF are ubiquitously expressed; and A-, C-, and H-MITF are enriched in the RPE (5,12,25–30). D-MITF is also expressed in RPE cells, and can also be detected in mac-
rophages and osteoclasts (31). E- and Mc-MITF are expressed in mast cells (18,32), and M-MITF is probably exclusive to melanocytes and melanoma (29,33), although it has been suggested that M-MITF may be found in osteoclasts (19) and mast cells (32).
In addition to the various MITF isoforms, two alternative splice forms have been discovered, which include or exclude six amino acids located near the basic region (5,12). This isoform difference arises from an alternative splice acceptor choice, and the isoform that includes the six amino acids is absent, because of a splice-site mutation, in the mouse mutant Mitfsp (12). B-, H-, and M-MITF have been shown to be expressed containing or lacking the six amino acids, however the existence of these splice vari- ants in other isoforms, and the functional significance of these six amino acids remains uncertain (8,24,25). Because of the complexity of the MITF locus, this chapter will focus on the melanocyte-specific isoform, M-MITF.
MITF AND WAARDENBURG SYNDROME
Waardenburg Syndrome (WS) is a hereditary condition characterized by deficiency of melanocytes in the skin, hair, eyes, and inner ear, resulting in hypopigmentation and deafness. Clinically, WS is divided into four subtypes (WS1–4) according to clinical features beyond the core melanocyte-related abnormalities. The core syndrome is seen in the WS2 subtype (34). Autosomal-dominant mutations of MITF result in WS2a and arise as a result of haploinsufficiency of the normal MITF allele (13,35,36). Conversely, dominant-negative MITF alleles appear to produce the more severe, though highly related, albinism-deafness disorder, Tietz syndrome (37,38).
Other subtypes of WS are characterized by abnormalities in addition to the features of WS2a. WS1 is associated with craniofacial deformities and WS3 (Waardenburg- Klein Syndrome) is associated with limb malformations. Both of these WS subtypes are caused by mutations in PAX3 (39–41), a transcription factor that is important both in melanocytes and in other developmental lineages, such as muscle. In Waardenburg- Shah Syndrome 4 (WS4 or Waardenburg-Hirschsprung disease), additional neurologi- cal symptoms, including megacolon are observed, caused by defective enteric ganglia, derivatives of the embryonic neural crest (42). WS4 is alternatively inherited in an autosomal recessive manner when associated with mutations of the endothelin-B recep- tor (EDNRB) (43) or its ligand, endothelin-3 (EDN3) (44,45), or as an autosomal domi- nant trait when SOX10 is mutated (42,46,47).
TRANSCRIPTIONAL REGULATION OF M-MITF
The regulation of M-MITF gene transcription appears to involve the interaction of multiple transcription factors that help confer the cell-type specific expression operating in the melanocyte lineage. The phenotypic convergence of WS2 with WS1, WS3, and WS4 has supported biochemical evidence that SOX10 and PAX3 are upstream regula- tors of MITF expression in melanocytes. Here, we will concentrate on the regulation of M-MITF by these, as well as by CREB and LEF-1/TCF transcription factors, shown to directly bind cis-acting consensus sequences and transactivate the M-MITF promoter/
enhancer. Several transcription factors have been shown to bind and regulate the M-MITF promoter (see Fig. 1), and it is plausible that others will be discovered, because the M-MITF promoter/enhancer contains several evolutionarily conserved binding sites that have yet to be validated.
Fig. 1. Transcriptional regulation of the M-MITF promoter. The M-MITF promoter is regulated by the transcription factors CREB, LEF-1/TCF, SOX10, and PAX3. After binding of D-melano- cyte stimulating hormone (D-MSH) to the melanocortin 1 receptor (MC1R), elevated cAMP levels produced by adenylate cyclase lead to phosphorylation of CREB, which binds cAMP- Responsive Element (CRE) to initiate M-MITF transcription. LEF-1/TCF-induced transcription depends on ligand (such as WNT1 or WNT3) binding to the Frizzled receptor, resulting in E-catenin release from its inhibiting complex, translocation to the nucleus, and activation of LEF-1/TCF. Although the signaling pathways affecting SOX10 and PAX3 are poorly understood, PAX3 can be affected by activated interleukin 6 receptor. Mutations in PAX3 and SOX10, as well as MITF, are associated with subtypes of the deafness-pigmentation disorder, Waardenburg Syndrome (WS), as indicated. (Graphical icons were adapted from www.biocarta.com.)
Regulation of M-MITF by CREB
M-MITF is a mediator of the pigmentation response in melanocytes, in which it is thought to bind and modulate the promoters of several major pigment enzymes via conserved E-box elements (15,48,49) in response to various stimuli, including the 12-amino acid peptide, D-melanocyte-stimulating hormone (D-MSH) (50,51).D-MSH binds to the seven-transmembrane Melanocortin 1 receptor (MC1R), which signals to increase the intracellular cyclic adenosine monophosphate (cAMP) (52). The elevated cAMP leads to phosphorylation and activation of the bZip factors CREB and ATF1 (53). Activated CREB/ATF1 can bind to a consensus cAMP response element (CRE) in the promoter of its target genes.
A CRE site is located at –147, relative to the transcription start site, in the promoter of M-MITF (8,50,54), and M-MITF expression can be induced via the D-MSH pathway and subsequently inhibited by an antagonist of MC1R (50,51,55). This cAMP response of M-MITF through CREB/ATF1 likely explains the observed cAMP responsiveness of M-MITF target genes, such as tyrosinase (Tyr). The cAMP response can also be activated with the application of various pharmaceuticals, such as forskolin, isobutyl- methylxanthine, and cholera toxin, circumventing the need for D-MSH stimulation (56).
With the addition of forskolin, MITF levels (RNA and protein) increase in a melanocyte- specific context (51). However, because activation of CREB/ATF1 can be induced by various pathways in multiple cell lineages, an important question would be how expres- sion of M-MITF is restricted to melanocytes. Recent data suggest that the specificity might be caused, at least in part, by cooperation between CREB/ATF1 and restricted SOX10 expression (57).
Regulation by SOX10
SOX10 is a member of the SRY box-containing (SOX) high-mobility group (HMG) transcription factor family that is expressed in neural crest derivatives (58). The study of the Dom (Dominant megacolon) mutant mouse which has Sox10 mutations (59), and the identification of WS4 patients with SOX10 mutations (42,60), helped identify SOX10 as a possible regulator of MITF. In situ hybridization using Dom mice, in which Sox10 is ablated, has revealed disturbances of Mitf expression (61). Although Sox10 is not required for proper development or early migration of the neural crest, postmigratory neural crest cells in mice lacking Sox10 undergo apoptosis before differentiation (62).
SOX10 does play an important role in melanocyte differentiation as a direct regulator of M-MITF (61,63–65). Several SOX10 consensus binding elements are present in the M-MITF promoter (61,63–65), and an additional two are present in the M-MITF distal enhancer (66). Transactivation of the M-MITF promoter by SOX10 can be attenuated by constructs manifesting SOX10 mutations identified in WS4 patients (63–65). The transactivation can also be amplified by addition of PAX3, suggesting a possible synergy with SOX10 (61,64,67). In addition, as stated in the previous paragraph, SOX10 has also been suggested to synergize with CREB/ATF1 to permit cAMP responsiveness of the M-MITF promoter in melanocytes (57).
Regulation by PAX3
Mutations in PAX3, a paired homeodomain factor, have been associated with WS1 and WS3 (40,41,67,68). WS1 patients have symptoms that overlap with WS2a patients, caused by mutation of MITF, consistent with a possible epistatic relationship of PAX-
3 to MITF (69). The M-MITF promoter has been shown to be regulated by PAX3 in melanoblasts, melanocytes, and melanoma cell lines (67,70). This transactivation failed when a melanoma cell line was transfected with constructs harboring PAX3 mutations, as identified in WS1 patients (67). The regulation of Mitf by Pax3 in mice seems to be further illustrated by the loss of melanocytes in the splotch mice, which harbor a Pax3 mutation (8,71). The role of Pax3 in the regulation of Mitf is further suggested by a study of B16/F10.9 melanoma cells, in which signaling via an interleukin-6 receptor/
interleukin-6 (IL6R/IL6) chimera led to a decrease in Pax3 mRNA and protein. This decrease was associated with downregulation of M-MITF mRNA and suppression of melanogenesis (72).
Regulation by LEF-1/TCF
LEF-1/TCF, HMG-domain transcription factors, are nuclear proteins that mediate WNT signaling via an interaction with E-catenin (73–75). The WNT proteins are secreted cysteine-rich glycoproteins that usually act in a paracrine fashion as the ligand for a seven-transmembrane Frizzled receptor. In the absence of WNT signals, E-catenin is complexed with the adenomatous polyposis coli tumor suppressor, axin, and glycogen synthase kinase-3E (GSK-3E). In this complex, GSK-3E phosphorylates E-catenin, thereby marking it for ubiquitination and subsequent degradation (73). In the presence of WNT signals, the canonical pathway allows for the accumulation of E-catenin by inhibiting GSK-3E. The accumulated E-catenin migrates to the nucleus, where it func- tions as a coactivator for DNA binding factors of the LEF-1/TCF family and subse- quently activates expression of target genes (76–78).
The WNT-signaling pathway has been shown to play a role in the expansion (79,80) and differentiation of neural crest cells (81–83). M-MITF is one of the targets of the WNT pathway as a downstream effector of WNT1 and WNT3a (23,84–86). Wnt1 and Wnt3a appear to have redundant roles in melanocytes, as demonstrated by loss of melanocytes in Wnt1–/–/Wnt3a–/– mice (80), whereas mutations in either one alone did not affect melanocytes (87,88). The M-MITF promoter contains LEF-1/TCF consensus binding sites (23,84) and is directly regulated by WNT/E-catenin when complexed with LEF-1, thereby activating the M-MITF promoter in mammals as well as in zebrafish (84,85,89).
The role of the WNT/MITF-signaling pathway in melanomagenesis is still being elucidated. Mutations in E-catenin that produce its stabilization have been found in melanoma cell lines (90), but it is less clear whether these occur in primary melanoma.
Nonetheless, some primary melanoma have been shown to have an accumulation of E-catenin in the nucleus, despite the lack of intrinsic mutations, suggesting that its stabilization has been dysregulated by an as yet poorly understood mechanism (91).
Importantly, M-MITF can rescue growth suppression of melanoma cell lines after dis- ruption of the E-catenin/LEF-1/TCF complex (89).
CRITICAL ROLES OF MITF IN THE MELANOCYTE LINEAGE
In light of the pigmentation defects in Mitfmi/mi mutant mice and the melanocyte- specific expression of the M-MITF isoform, it was expected that MITF transcriptional targets would include genes affecting critical melanocyte functions, such as survival, proliferation, and differentiation. Indeed, data accumulating over the past decade have emphasized the importance of MITF in the melanocyte lineage, as a key regulator of various cellular processes.Melanocyte Survival
Functional Mitf protein is required for melanocyte development and maintenance.
Melanoblasts lacking Mitf die within 48 h of appearance (30), demonstrating its neces- sity for their survival. However, Mitf presence is critical not only for melanoblasts, but also for the continued existence of differentiated cells of the melanocyte lineage. Post- natal melanocyte death and premature graying were found in hypomorphic alleles of the microphthalmia gene, such as in the Mitfvit/vit mice, which are born with slightly lighter than normal coat color that turns white prematurely (12,92). A similar, although more accelerated, phenotype was observed with Bcl2 knockout mice, which turn gray with the second hair follicle cycle (93). This phenotype was reversed in 90% of Bim–/– Bcl2–/–
double knockouts, but not in Bim+/– Bcl2–/–mice, emphasizing the delicate balance between proapoptotic and antiapoptotic proteins in melanocytes (94). The identification of BCL2 as an MITF direct target gene in melanocytes (95) underscored the importance of MITF in melanocyte survival, and hinted that it might contribute to the notorious resistance of melanoma to traditional chemotherapeutic treatments.
Pigment Production
Pigment production in the melanocyte lineage takes place only after cellular differ- entiation of unpigmented melanoblasts to melanocytes. Among the markers of melano- cyte differentiation is the copper-binding protein tyrosinase (Tyr), is a melanosome membrane protein that catalyses the initial rate-limiting steps in the process of melanin biosynthesis, oxidizing tyrosine to dioxyphenylalanine (DOPA), and DOPA to DOPA quinone (reviewed in ref. 96). Mutations in the Tyr locus result in various forms of hypopigmentation/albinism in mice (e.g., albino, platinum/cp mutations, refs. 97–99), and are responsible for oculocutaneous albinism type I syndrome in humans, severely affecting pigmentation of the skin, hair, and eye (reviewed in ref.100). Expression of Tyr is restricted to melanocytes and RPE cells via a proximal promoter (reviewed in ref. 101), and is probably amplified by an upstream distal enhancer (49,102–104). The identifica- tion of Tyrp1 and dopachrome tautomerase (Dct)/Tyrp2, tyr-related genes involved in later steps of melanin synthesis (105–107), enabled analyses of promoter sequences, in a search for common elements that would account for the spatiotemporal expression of pigment-producing enzymes (108–110). Detection of conserved DNA sequence elements in the promoters of all three genes, along with the finding that Mitf is capable of binding and transactivating this sequence element in vitro (15), lead to the hypothesis that Mitf might regulate the expression of melanin biosynthesis enzymes. Indeed, reporter assays have demonstrated the ability of Mitf to transactivate the promoters of all three Tyr family enzymes, in an E-box-dependent manner (15,48,49,111–114). In addition, inhibition of Mitf activity downregulated Tyr and Tyrp1 transcription (115,116), strengthening the upstream role of Mitf in regulation of pigmentation. Interest- ingly, overexpression of wild-type Mitf in either mouse B16 melanoma cells or human melanocytes did not upregulate Tyr levels, implying that Mitf alone is not sufficient to induce Tyr expression and might depend on cooperating protein(s) (116). It is possible that a cooperative mechanism will also be implicated in the regulation of the DCT pro- moter. Although MITF can drive transactivation of a reporter gene from the DCT pro- moter (111,114), a microarray screen for MITF target genes in melanocytes found that after stem cell factor (SCF) or 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulation, DCT expression was cycloheximide-sensitive and MITF independent (95). DCT is also
expressed in the RPE cells of homozygous MITFvga-9mice, although the vga-9 mutation behaves like a null allele (5,30). These data point to combinatorial regulation, and, indeed, recent studies reported synergism between MITF and LEF-1 (117) or SOX10 (114) in DCT/TYRP2 reporter assays. Undoubtedly, MITF expression is crucial for pigment production, yet the exact mechanisms by which Mitf regulates pigmentation are still under investigation.
Additional MITF Target Genes
As a protein with restricted expression, MITF is a clinically important diagnostic marker for melanocytic neoplasms, including melanoma (118–120). Recently, two other commonly used melanoma markers, SILV/Pmel17/GP100 and MLANA/MART1, were found to be regulated by MITF (121). SILV is a melanosomal matrix protein, and although its function is still unknown, an insertion altering its C-terminus induces the silver coat color phenotype in mice (122,123). MLANA/MART1 is a membrane protein whose expression is restricted to melanocytes and RPE cells, and has been suggested to play a role in early melanosome biosynthesis (124,125). Expression levels of MITF, SILV, and MLANA/MART1 were found to be tightly correlated both in primary melanoma and melanoma cell lines, collectively representing the differentiation level of the tumor cells (121).
Another melanocyte differentiation antigen regulated by MITF is MATP/AIM1 (126), a potential sucrose transporter. Mutations in the mouse Matp gene are the cause of the underwhite phenotype, exhibiting fur and eye hypopigmentation, sometimes in an age- dependent fashion (127). MATP is evolutionarily conserved, and a mutation in the medaka fish homolog, designated b, causes melanin loss and results in a goldfish-like phenotype caused by default nonmelanin pigments (128). Interestingly, MATP/AIM1 mutations were found in several oculocutaneous albinism type IV patients (see OMIM:606202), but their exact contribution to the phenotype is unknown.
Recently, MITF was found to be an important regulator of TRPM1/Melastatin1, a potential calcium channel protein (129). Expression levels of TRPM1 inversely corre- late with melanoma metastasis (130), although the functional role and importance of TRPM1 are still enigmatic.
As the directory of MITF target genes is constantly growing, it is important to differ- entiate between immediate MITF-responsive promoters and secondarily affected genes.
Several key transcription regulators were suggested to be either MITF targets or cyclo- heximide-resistant c-Kit/TPA and MITF-dependent targets in melanocytes, including the AP-1 subunits c-FOS and JUNB, ATF4, ELK1, and TBX2 (95). It is plausible that the wide effect of MITF on the melanocyte lineage is at least partially mediated by regulation of such transcription factors. Although additional experiments are needed to verify the effect of MITF on the former ones, Tbx2 levels were shown to be regulated by Mitf in vitro, and Tbx2 is absent from Mitf-negative melanoblast precursor cells (131). Acting as a transcriptional repressor, Tbx2 was found to negatively regulate the Tyrp1 promoter (132). However, because Tbx2 has been implicated in suppression of cell senescence through downregulation of Cdkn2a (p19ARF) (133) and Cdkn1a (p21WAF1) (134), its effects in melanocytes might also be cell-cycle related.
An additional transcriptional repressor whose promoter was found to be regulated by MITF in vitro is SNAI2/Slug (135). Like MITF, SNAI2 can bind E-box motifs, and mutations in SNAI2 also result in melanocyte loss in human and mouse (135,136). In this
regard, the ability of SNAI2 to promote cell survival (137) and to downregulate E-cadherin expression in breast cancer (138) might suggest a role in melanoblast survival and/or migration, respectively, downstream of MITF. It is virtually certain that MITF is involved in regulation of additional target genes in melanocytes. Further identification of these targets may provide important clues to the key functional role for MITF in the melanocyte lineage.
POSTTRANSLATIONAL MODIFICATIONS OF MITF
As a key regulator of melanocyte processes, control of MITF expression and activity occurs at several levels, in addition to the transcriptional level described above. Although regulation of MITF RNA stability is poorly understood, posttranslational modifications control MITF transactivation capacity and stability (seeFig. 2). The phenotypic similari- ties between mutations in the c-Kit receptor tyrosine kinase, its ligand Kitl/SCF, and Mitf (described in 139), suggested that the Mitf protein might be regulated by Kit-dependent phosphorylation. Biochemical analysis led to characterization of phosphorylation sites controlling the transactivation activity of MITF, and its half-life (140). On activation of the Kit/MAPK pathway, MITF is phosphorylated on Ser73 by MAPK1 (ERK2) (140).
This modification allows it to recruit the transcriptional coactivator p300/CBP (141).
MITF transactivation capacity may be further regulated by additional phosphorylation on Ser409 by the RPS6KA1 (RSK1) kinase (a direct target of ERK), which leads to ubiquitin-mediated proteolysis (142). Lysine 201 of MITF appears to be a potential ubiqitination site, because mutating it to alanine blocked proteasomal degradation of MITF (143). Activity of MITF seems to be dependent also on Ser298, positioned down- stream of the bHLH-LZ domain. In vitro phosphorylation of Ser298 by GSK-3E was seen to enhance MITF transcriptional activity in reporter assays (144). The physiologi- cal importance of this residue is emphasized by characterization of a Ser298 mutation in individuals with WS2 (39). Although not reported in melanocytes, phosphorylation of MITF on Ser307 was found in osteoclasts after RANKL stimulation, in a p38-depen- dent manner (145). It is possible that the MITF protein is further modified on additional residues by various signaling pathways, yet to be found.
PROTEIN–PROTEIN INTERACTIONS OF MITF
In addition to its ability to form heterodimers with other MiT family members (TFE3, TFEB, and TFEC), MITF was found to interact with several other proteins, including transcription factors. As a suggested mechanism of self-amplification in melanocytes, MITF was reported to bind LEF-1, and to recruit it to its own promoter (146). In osteo- clasts, MITF was reported to bind PU.1, an Ets family transcription factor expressed in hematopoietic cells (147). MITF acts downstream of PU.1 during osteoclast develop- ment, and mutations in PU.1 or MITF can cause osteopetrosis (reviewed in ref. 148). An additional transcription factor that was shown to interact with MITF is Pax-6 (149).
Because both Mitf mutations and Pax-6 mutations cause eye abnormalities, this interac- tion might be important for normal RPE differentiation in vivo (149).
A yeast two-hybrid screen for MITF binding proteins identified UBC9, an E2 SUMO ligase, which was suggested to regulate MITF degradation through the ubiquitin- proteasome system (143). Interestingly, identification of an MITF interaction with PIAS3, an E3 SUMO ligase, suggests that an effect of UBC9 on MITF might be SUMO-
Fig. 2. Regulation of the MITF protein. After activation of the MAPK pathway, following c-KIT receptor binding by its ligand stem cell factor (SCF), MITF is phosphorylated at Ser73 by ERK2.
p90RSK, acting downstream from activated ERK1/2, induces a second MITF phosphorylation, on Ser409. These modifications activate MITF, which, together with the coactivators p300/CBP, initiates transcription of target genes from E-Box elements. However, phosphorylation on Ser73 also sensitizes the protein to degradation by the proteasome via ubiquitination of Lys201. GSK-3E, acting downstream of the WNT/AKT pathways can phosphorylate MITF on Ser298. (Graphical icons were adapted from www.biocarta.com.)
mediated (150,151). PIAS3 was found to downregulate the transcriptional activity of MITF, but MAPK/RSK1 phosphorylation on Ser409 of MITF disrupted PIAS3 binding and enhanced MITF transactivation capacity (151). Another potential inhibitor of MITF is PKCI/HINT1, which is thought to regulate MITF in mast cells (reviewed in ref. 152).
Embryonic fibroblasts from PKCI null mice show cell-cycle irregularities and increased
resistance to cytotoxicity by ionizing radiation, although the mice themselves have no noticeable phenotype (153). Analogous to the MITF inhibition by PIAS3, MAPK- induced phosphorylation of MITF releases PKCI inhibition (154).
MITF IN CANCER
A recurrent theme in cancer is the dysregulation of transcription factors. As effectors of multiple downstream genes, transcription factor levels and function are tightly regu- lated. However, when mutated or abnormally expressed, these proteins might lead to severe consequences, such as cancer. A well-known example is the MYC proto- oncogene, a bHLH-LZ protein that heterodimerizes with MAX to bind E-box sequences.
Dysregulated expression of c-MYC is found in numerous cancer types, because of gene amplification or chromosomal translocation. Data emerging over the past few years have suggested that the MiT family of transcription factors might be involved in malignancy.
Chromosomal translocations of TFE3 were found in Papillary Renal Cell Carcinoma and in Alveolar Soft Part Sarcomas (reviewed in ref. 155). In these instances, the bHLH-LZ domain of TFE3 was found fused downstream from a variety of different donor proteins, in each case forming a chimeric protein that retains the DNA-binding capacity of TFE3.
Another mechanistically informative pathogenic event involves the recent identification of TFEB translocations in papillary renal cell carcinomas (156–158). In these particular cases, a translocation was observed between chromosomes 6 and 11, and SILV/Pmel17/
GP100 expression was observed in the tumor. The expression of this MITF target sug- gested the possibility that TFEB (located on chromosome 6) may be present at the translocation breakpoint. In this case, the translocation placed the entire open reading frame of TFEB downstream of the breakpoint, and the alpha gene into which TFEB was inserted contributed no open reading frame to the resulting transcript (158). This trans- location thus represents a pure example of promoter swapping and strongly suggests that the common mechanistic event in the MiT family translocations is dysregulated expres- sion of a potentially normal MiT transcription factor (157,158). Similar promoter sub- stitution was shown to be the mechanism for c-MYC-induced Burkitt’s lymphoma (159,160). Considering the MiT family’s capacity to regulate transcription via E-box elements similar to c-MYC, it is possible that a subset of c-MYC target genes may also be regulated by MiT proteins, and participate in cellular transformation, although this remains to be determined.
Although chromosomal abnormalities of the MITF gene have not been reported, MITF has emerged as a clinically useful histopathological marker for melanocytic lesions and has become incorporated as a common immunohistochemical target in melanoma diagnosis (118,161–163). Interestingly, MITF levels often decrease during melanoma progression, although MITF signal has been detected in virtually all samples examined including amelanotic melanoma (118,161–163). It is notable that expression of MITF targets such as TRPM1/melastatin1, HMB/pmel17/silver, and MLANA/Mart1 has also been seen to diminish in association with melanoma progression. The reason for this is unknown, but one may speculate that diminished MITF expression would be associated with a less differentiated phenotype (e.g., suppression of pigmentation), which may provide a growth advantage. Nonetheless, the observation that measurable MITF ex- pression is usually retained may suggest that different target genes participate in the survival role of MITF as compared with differentiation/pigmentation, and that, mecha-
nistically, these target genes might vary in the dose of MITF required for their regulation.
Although the role of MITF in melanoma is certainly far from clear at this time, its ability to upregulate BCL2 (95), an antiapoptotic gene, may be of particular importance. More- over, a recent report showing cooperation between MITF and constitutively activated STAT3 in transformation of NIH-3T3 cells (164) points to a potential contribution of MITF in transformation, perhaps via upregulation of downstream targets, such as c-Fos (95,164). The emerging picture from these data points to critical regulation of cellular survival and proliferation by MiT transcription factors.
CONCLUSIONS AND PERSPECTIVES
MITF is a remarkable factor in serving as a key master regulator of the entire neural crest-derived melanocyte lineage. Its loss of function produces dramatic phenotypes of melanocyte loss in numerous species, including humans. Furthermore, gain of function or dysregulated expression of its family members appear to be oncogenic in certain cell lineages. MITF plays a pivotal role in differentiation of melanocytes, largely through its ability to regulate pigment enzyme and processing gene expression. Yet, MITF clearly regulates other target genes, because pigmentation gene loss presumably cannot account for lack of melanocyte survival, as albino melanocytes are highly viable. The search for a more complete list of MITF transcriptional target genes, coupled to increased under- standing of the signaling pathways and environmental cues that regulate MITF pretranslationally and posttranslationally, will stand to provide crucial information that may be of importance in both benign conditions (affecting pigmentation and deafness) as well as a variety of human malignancies, including melanoma and other MiT family- associated cancers.
ACKNOWLEDGMENTS
The authors acknowledge the outstanding contributions of numerous investigators to the work presented in this chapter. We also gratefully acknowledge the grant support of the National Institutes of Health, and the valuable advice and discussions from all mem- bers of the Fisher lab, past and present. D. E. F. is Jan and Charles Nirenberg Fellow in Pediatric Oncology at the Dana-Farber Cancer Institute.
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