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Keywords:

  • Microphthalmia-associated transcription factor;
  • Brn-2;
  • Tbx2;
  • Melanocytes;
  • Melanoma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

The enormous variety of pigmentation phenotypes in nature reflects a series of remarkable events that begin in the neural crest and end with the manufacture and distribution of pigment by mature melanocytes located in the epidermis and hair follicles. While the origins of melanoblasts from multipotent precursors in the neural crest is striking in itself, yet more so is the fact that these pioneer melanoblasts manage to undertake and survive their long migration, and in doing so proliferate and maintain their identity before ultimately arriving at their destination and undergoing differentiation. With the application of the powerful combination of genetics and molecular and cell biology the mystery surrounding the genesis of the melanocyte lineage is slowly being unravelled. At its heart is the powerful alliance between signal transduction and transcription that coordinates the program of gene expression that confers on a cell its identity, provides its passport for migration, and instructs it in the arts of survival and timely reproduction. The realization that the proliferation and migration of melanoblasts during development resembles closely the proliferation and metastasis of melanoma, a highly dangerous and increasingly common cancer, serves to highlight the value of the melanocyte system as a model for addressing key issues of general significance in both development and cancer.


Abbreviations – 
bHLH-LZ

basic-helix–loop–helix-leucine zipper

ERK2

extracellular signal-regulated kinase 2

GSK3β

glycogen synthase kinase 3-β-kinase

IL-6

interleukin-6

ITF2

initiation transcription factor 2

Lef1

lymphoid enhancer factor 1

MAP kinase

mitogen-activated protein kinase

Mitf

microphthalmia-associated transcription factor

Tcf

T-cell factor

The Center of the Network – Microphthalmia-Associated Transcription Factor

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

The discovery 10 yr ago of the gene encoding the basic helix–loop–helix leucine zipper (HLH-LZ) microphthalmia-associated transcription factor gene (Mitf) (1, 2), provided a major impetus to the study of transcription regulation in the melanocyte lineage. Indeed, Mitf appears to be at the heart of a regulatory network of transcription factors and signalling pathways that control the survival, proliferation and differentiation of melanoblasts and melanocytes. The interest generated by Mitf is reflected in the publication of a number of recent reviews that detail its regulation and function (3–6) and it will again be subject to intense scrutiny in an upcoming review by E. Steingrímsson, N.G. Copeland, N.A. Jenkins (unpublished data). Given the amount of information already available it is perhaps better to summarize briefly those aspects of the melanocyte-specific isoform of Mitf, Mitf-M, that are now well established while discussing in more detail the progress made in understanding the function of this enigmatic transcription factor.

Mitf Expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

The Mitf gene is absolutely required for the generation of mature pigment cells and as a result many different alleles of Mitf have been described that affect Mitf function and which consequently lead to reduced numbers of melanocytes (6–8). The expression of mouse and human Mitf is controlled by several promoters that specify expression patterns (9). The best characterized is the Mitf-M promoter (10) that is essential for Mitf expression in the neural crest-derived melanocyte population. Mitf is one of the earliest markers of the melanocyte lineage and expression of the Mitf-M gene can be activated by an array of transcription factors that bind its promoter (Fig. 1A). These include the paired homeodomain factor Pax3 (11) that can also regulate the Tyrp1 promoter (12). The key role of Pax3 in regulating Mitf expression has also been highlighted by a recent study that implicated a loss of Pax3 RNA and protein expression in the down-regulation of Mitf as a consequence of signalling from the interleukin-6 (IL-6) receptor in B16 melanoma cells (13). The results clearly linked IL6 signalling to the transcription factor STAT3, but how STAT3 mediated the down-regulation of Pax3 expression was not clear. Indeed the role of the STAT family of transcription regulators in melanocyte development or differentiation has yet to be explored systematically. Nevertheless increasing evidence points to a potentially important role in melanoma with inhibition of STAT3 function leading to reduced growth and increased apoptosis of B16 melanoma cells (14, 15) and aberrant nuclear accumulation of STAT5 in human melanomas (C. Wellbrock, personal communication). Interestingly, Mitf appears to cooperate with STAT3 in transformation of 3T3 cells and up-regulation of Fos expression (16), a target also identified in the gene array screen for genes up-regulated by Mitf (17).

image

Figure 1. Regulation of Mitf expression and activity. (A) The Mitf-M promoter showing the binding sites for transcription factors that are known to regulate Mitf expression and the signal transduction pathways regulating their activity. (B) Features of the Mitf protein. Phosphorylation sites and locations of regions interacting with co-factors or other transcription factors are indicated above, while those genes that are known to be regulated by Mitf are shown below.

Download figure to PowerPoint

The HMG box protein Sox10 binds both the proximal Mitf-M promoter (18–21) as well as an upstream enhancer (22) and can cooperate with, although not interact with, Mitf in activation of the dopachrome tautomerase (Dct) promoter (23, 24). Indeed genetic evidence suggests that the principle function of Sox10 in the melanocyte lineage appears, at least in Zebra fish, to be in activation of Mitf expression (25). The role of Sox10 and Pax3 in regulating Mitf expression is underpinned by the observation that mutations in either Pax3 or Sox10 are genetically implicated in Waardenburg syndrome type 1 (26–28) and Waardenburg–Hirschsprung disease (18, 29) respectively, human syndromes characterized by pigmentation defects that may be explained if these factors are necessary for inducing or maintaining the correct level of Mitf expression.

The lymphoid enhancer factor/T-cell factor (Lef1/Tcf) family of transcription factors that interact with β-catenin enable Mitf expression to be regulated by Wnt signalling (30–33). The regulation of the Mitf promoter by Wnt signalling via a Lef1/Tcf binding site (30, 31) most likely explains why over-expression of components of the Wnt signalling pathway promotes an increase in the numbers of melanoblasts in the neural crest (34, 35) and why mice lacking both Wnt-1 and Wnt-3a lack Dct-positive cells migrating from the neural crest at embryonic day 11.5 (E11.5) (36). While Wnt/β-catenin signalling can activate the Mitf promoter through Lef1/Tcf factors, the bHLH-LZ domain of Mitf can interact with Lef1 but not with the related factor Tcf1 (37). One consequence of the interaction is that Mitf and Lef1 can synergistically activate the DCT promoter. Similar results were also obtained with the Mitf-related factors TFE3 and TFEC but not with Myc. In support of a role for Lef1–Mitf interaction in vivo, Lef1−/− mice contain unpigmented melanocytes in their skin while exhibiting no apparent defects in the melanocyte lineage during development (38). It is possible therefore that another member of the Lef1/Tcf family is responsible for activation of the Mitf-M promoter by Wnt/β-catenin signalling during development but that Lef1–Mitf interactions make a significant contribution to transcription in postnatal melanocytes.

The bZip factor CREB and related factors confer on the Mitf-M promoter responsiveness to cAMP and melanocortin-1 receptor (MC1R) signalling (39, 40) and therefore indirectly account for the cAMP-responsiveness of Mitf target genes. One intriguing observation is that the cAMP-responsiveness of the promoter appears to be cell type-specific despite the fact that CREB and other transcription factors able to mediate the cAMP response are ubiquitously expressed (41). Two possible explanations present themselves. First it is possible that the core promoter of Mitf is recognized by a cell type-specific basal transcription complex. Cell type-specific TBP-associated factors (TAFs) have been observed in other cell types (42) and the Tyrp-1 promoter TATA region contributes substantially to melanocyte-specific gene expression (43). A second, and not mutually exclusive possibility is that the ability to activate the Mitf promoter by cAMP will depend on at least some basal expression of the promoter prior to the cAMP signalling pathway being activated. In support of this model Huber et al. (44) recently demonstrated that the Mitf promoter was inactive and refractive to cAMP signalling in a neuroblastoma cell line, but that ectopic expression of Sox10 in these cells could restore the cAMP-responsiveness of the promoter. Whether Sox10 is the only factor with the capacity to cooperate with cAMP-responsive transcription factors at the Mitf promoter is not known, although it has been discussed (44) that mutating the Pax3 and Lef1 binding sites in the Mitf promoter do not affect its ability to respond to cAMP.

In addition, the homeo domain factor one-cut (45) can activate the Mitf promoter, at least in transfection assays although there is no genetic evidence to underpin its importance. It is also likely, although has not been formally shown, that the bHLH factor initiation transcription factor 2 (ITF2) whose expression is repressed by cAMP (46) may also act to repress Mitf expression and thereby indirectly regulate melanogenic genes. One reason for such a variety of transcription factors regulating the melanocyte-specific Mitf promoter would be to facilitate tight control and responsiveness of the Mitf promoter to a wide range of signals that control melanocyte/melanoblast survival, proliferation, migration and function. However, how factors recognizing the Mitf promoter cooperate to activate Mitf expression in a highly specific subset of neural crest cells is yet to be understood.

Regulation of Mitf

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

The notion that Mitf protein and RNA levels are important is reinforced by the fact that Mitf protein levels are regulated by mitogen-activated protein kinase (MAP kinase) signalling downstream of receptor tyrosine kinases such as Kit which is also required for melanocyte development: phosphorylation of Mitf at Ser73 by the MAP kinase extracellular signal-regulated kinase 2 (ERK2) and at Ser409 by the MAP kinase-activated kinase RSK leads to its ubiquitination and degradation (47–49), at least in cells in culture (Fig. 1B). While it is evident that MAP kinase signalling is a key lever on Mitf stability, it is still unclear when during development Mitf phosphorylation takes place and whether phosphorylation on Ser73 and Ser409 always occur together or whether they may be uncoupled. In this respect, it is important to note that the kinetics of MAP kinase activation are not always the same as that of RSK, despite the fact that RSK activation is dependent on MAP kinase mediated phosphorylation (50). In addition to regulating Mitf stability it has also been proposed that MAP kinase-mediated phosphorylation of Mitf promotes the interaction with the CBP/p300 transcription cofactor that via a classical acidic amphipathic helical trans-activation domain located N-terminal to the bHLH-LZ DNA binding and dimerization domain (51, 52). At the same time, phosphorylation of Ser409 by RSK has also been implicated in the inhibition of an interaction between the Mitf Zip domain and PIAS3, a co-immunoprecipitating factor identified initially as interacting with Mitf in a 2-hybrid screen. Interaction with PIAS3 can severely inhibit the ability of Mitf to bind DNA and activate transcription (53, 54). Intriguingly PIAS proteins, including PIAS3, interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9 (55). Ubc9 was previously identified as interacting with Mitf and participating in its ubiquitination and proteosome-mediated degradation (49). However, the identification of PIAS3 as an interacting protein may suggest Mitf may also be sumolyated and raise the possibility that sumolyation and ubiquitination may be antagonistic events. In any case, phosphorylation on Ser73 and Ser409 is likely to lead to a transient increase in transcription activation by promoting Mitf interaction with CBP as well as release of PIAS3 and increased DNA-binding before triggering subsequent degradation of Mitf.

DNA binding by Mitf also seems to be promoted by phosphorylation on Ser298 by glycogen synthase kinase3β (GSK3β) lying downstream from AKT and PI3K, but not by GSK3 β participating in the Wnt/β-catenin cascade (56). The importance of Ser298 in Mitf function in vivo is highlighted by the fact that it is mutated in Waardenburg syndrome type 2 (57), although it is unclear whether any defect in a Ser298 mutant arises because of structural difference in the protein as opposed to an inability of the mutated residue to be phosphorylated.

Mitf Targets and a Possible Role as a Negative Regulator of Proliferation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

In vivo Mitf recognizes a specific subset of so-called E-box motifs characterized by a core CATGTG sequence flanked by either a 5’T, 3’A, or both and the related M-box in which additional flanking bases are conserved (58). Full consensus Mitf-targets have been identified in the promoters for a wide range of genes regulated in the melanocyte lineage including Tyrosinase, Tyrp1, DCT [reviewed in (3)], Silver (59) and the QNR71 (60) genes, all of which are implicated in melanosome function, as well as the melanocortin 1 receptor (61, 62). The identity of these target genes would place Mitf as a factor implicated in melanocyte differentiation by regulating genes involved in melanosome function. In humans, the tanning response affords protection from UV-mediated skin damage and is characterized by increased production of melanin and its transfer from melanocytes to surrounding keratinocytes. UV-mediated activation of stress signalling pathways can lead to increased transcription of genes such as tyrosinase (63) encoding the rate limiting enzyme in the manufacture of pigment. While mitogenic signals appear to decrease Mitf protein levels, recent evidence obtained using osteoclasts and a phosphorylation site-specific antibody suggest that stress signalling via the p38 kinase results a rapid and persistent phosphorylation of Mitf on Ser307 and leading to an increased ability of Mitf to activate transcription (64). It seems likely that regulation of Mitf by p38 will also feature in melanocytes and the ability of Mitf to be phosphorylated by p38 should in principle make it responsive to UV, although this has yet to be tested. Any UV-mediated activation of Mitf would in turn have implications for our understanding of how UV induces transcription of the tyrosinase promoter.

Previous work identified the ubiquitous bHLH-LZ transcription factor Usf-1 both as a target for the p38 kinase and as a factor necessary for the UV-responsiveness of tyrosinase expression through its ability to bind the M-box and related E-box elements in the promoter in vitro and in vivo (63). As both Mitf and Usf-1 can bind the same sequences but cannot bind the same element simultaneously, the observations that Mitf and Usf-1 are both activated by p38 potentially raises an interesting question concerning the relative role of each factor at the tyrosinase promoter, namely do these proteins co-operate at tyrosinase and if so do they bind the promoter simultaneously with each factor recognizing a different E box in vivo, or sequentially, with one factor binding the promoter at one time and being displaced by the other. At the moment it is not possible to distinguish between these possibilities, but sequential occupancy of a single E box by bHLH (LZ) factors has been described in yeast where the bHLH-LZ factor Cbf1 is required to maintain the correct chromatin structure at the repressed PHO8 promoter and is displaced by the bHLH transcription activator Pho4 when expression of the PHO8 gene is induced (65).

Our knowledge of the controls operating on the Mitf-M promoter and of the signalling pathways regulating Mitf protein stability or function in terms of regulating genes implicated in pigmentation has increased rapidly over the past few years. However, the role of Mitf early in development is not at all well understood. Clearly, the fact that Mitf-negative melanoblasts die within 2 d of their appearance (66) indicates that Mitf has a critical role in melanoblast survival. How Mitf exerts part of its survival function was partly revealed as a result of a microarray analysis of genes regulated by Mitf (17). One of the genes up-regulated by Mitf in this screen was BCL2, a gene known to play a critical role in inhibition of apoptosis. Bcl2-deficient mice exhibit a loss of melanocytes shortly after birth implicating Bcl2 in postnatal melanocyte survival (67, 68). In contrast Bcl2+/− animals exhibit a normal coat colour, but an Mitfvit/+, Bcl2+/− compound heterozygote is characterized by a gradual increase in melanocyte loss over time (17). These results indicate that Mitf and Bcl2 either work together in the same pathway or cooperate in parallel pathways to promote melanocyte survival. The case for the former possibility was strengthened by chromatin immunoprecipitation assays that indicated that Mitf was present at the Bcl2 promoter in the 501 mel human melanoma cell line (17). In addition, binding of Mitf to a CATGTG element in the promoter could be observed in vitro in the presence of anti-Mitf antibody, and mutation of the element prevented the activation of the promoter observed in cells infected with an Mitf expressing adenovirus. However, the CATGTG E-box present in the Bcl2 promoter does not fit the full consensus for Mitf binding described previously (58) and mutation of this element failed to decrease the activity of the Bcl2 promoter in 501 mel cells that contain active Mitf. It is possible though that Mitf gains access to this apparently low affinity site by cooperation with other factors within the context of the Bcl2 promoter.

Although Mitf regulation of Bcl2 may account in part for the role of Mitf in postnatal melanocyte survival and indeed in melanoma, it is also clear that other pathways must operate during development since Mitf−/− mice lack all pigment cells while Bcl2−/− animals are pigmented at birth. There is accumulating evidence, although much of it indirect, to suggest that Mitf may have a key role in controlling proliferation. Thus, the rate of melanoblast proliferation is increased in Mitfmi heterozygous mice (69); loss of Mitf results in hyperproliferation of the RPE (66) and mis-espression of Mitf in the presumptive retina correlates with reduced proliferation (70); Mitf can be activated by p38-mediated stress signalling, a negative influence on cell cycle progression (64), and degraded in response to mitogenic signalling via the MAP kinase pathway (48, 49); and repression of Mitf expression by a variety of oncogenes (71–73) leads to increased proliferation. Although none of these results alone provides sufficient grounds for believing in Mitf as a negative regulator of proliferation, together they make a sufficiently powerful argument that the possibility should be considered. Indeed, the ability of Mitf to promote melanoblast survival may be intimately related to its apparent ability to control cell division. One important point to note however is that if in fact Mitf does inhibit proliferation, mutations that mildly reduce Mitf function may give no pigmentation phenotype since the melanoblasts exiting the neural crest might survive less well but proliferate more rapidly leading to an normal number of mature melanocytes being present in the epidermis and hair follicles at birth.

While the ability of Mitf to regulate pigmentation genes may be important for its role in differentiation, targets that are likely to be implicated in other functions, such as cell cycle control or melanoblast migration would include the genes encoding the zinc-finger DNA-binding protein Slug (SNAI2) and the Tbx2 transcription factor. The Slug promoter is a direct target for Mitf and is genetically linked to Mitf since mutations in either Mitf or Slug (in addition to Pax3 and Sox10) can give rise to Waardenburg syndrome, characterized in part by the manifestation of pigmentation defects (74). In the mouse, the Slug gene, Slugh, is not essential for neural crest development (75) but is expressed in migratory neural crest cells and mice homozygous for Slug loss-of-function mutations can exhibit pigmentation defects characteristic of reduced melanocyte numbers (76). Precisely how Slug might affect the melanocyte lineage is not clear although its ability to repress E-cadherin expression may be important (77, 78). Thus Mitf-mediated regulation of Slug could play a role in orchestrating the correct expression of adhesion molecules in melanocytes or melanoblasts. Indeed there is accumulating evidence that Mitf may well play a key role in regulating dendrite formation and cytoskeletal architecture. For example, a melanoma cell line ectopically expressing Mitf-M was converted from an epitheloid to a spindle-cell type in vivo (79), while over-expression of Mitf in 3T3 cells can also lead to an increase in dendricity (80). Taken together the evidence suggests that Mitf might act as a regulator of melanocyte morphology and that this in turn may well be related to the ability of melanoblasts to migrate in vivo. Precisely which genes are involved in this process is currently not known but identifying the relevant genes is likely to represent fruitful areas for future research.

The Tbx2 transcription factor is a member of the T-box family of transcription factors whose members play critical roles in many aspects of development [see (81) for review]. Outside the melanocyte lineage Tbx2 has been reported to play a role in digit identity in the chick (82) and targeted disruption of Tbx2 in mice leads to embryonic lethality arising from heart defects (V.E. Papaioannou, personal communication). That the Tbx2 promoter is a bone fide target for Mitf comes from several lines of evidence: The Tbx2 promoter contains a TCATGTG motif that binds and is regulated by Mitf in vitro and in vivo (83); a gene array analysis of cells ectopically over-expressing Mitf identified Tbx2 as a direct target (17); and more recently ectopic expression of Mitf in Medaka embryonic stem cells led to a rapid induction of Tbx2 mRNA and the generation of melanocytes (84). In addition to its regulation by Mitf, Tbx2 is also a target for retinoic acid signalling in B16 melanoma cells (85). The precise role of Tbx2 in the melanocyte lineage is unclear although it was first identified as a repressor of the Tyrp-1 promoter by binding two conserved GTGTGA elements known as the MSEi and MSEu (43, 86). More recently however, Tbx2 has been shown to repress the expression of the p21Waf1 cyclin-dependent kinase inhibitor gene (CDKN1A) through a GTGTGA motif located at the p21 transcription initiator (87). The ability of Tbx2 to repress p21, as well as the p19ARF promoter (88, 89), is likely to be fundamental to its striking ability to suppress senescence (87–89). Possibly Mitf may use Tbx2 to control progress through the cell cycle or to prevent cells receiving a strong MAP kinase signal from undergoing senescence.

Melanoma

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

One impetus to investigating melanocyte development is the clues it gives to understanding melanoma, an increasingly common and highly aggressive cancer. Accumulating evidence would indicate that common pathways operate to control proliferation in melanoblasts and melanoma; melanoblasts proliferate and migrate whereas melanoma cells proliferate and metastasise. In addition to the loss of cell cycle regulators such as the CDKN2a (p16) locus, melanoma is been characterized by the constitutive activation of two signal transduction pathways: First, Wnt/β-catenin signalling is constitutively active in a significant minority of melanomas (90, 91). The importance of this pathway has been highlighted by the observation that in melanoma cells interference with the Wnt/β-catenin signalling pathway leads to suppression of clonogenic growth (92). This block to growth can be overcome by expression of Mitf that facilitates growth by a pro-survival mechanism (92). Second, activation of the MAP-kinase signalling pathway as a result of activating mutations in BRAF occur in around 70% or melanomas (93) and nevi (94) and further deregulation of MAP kinase signalling may be achieved through receptor tyrosinase kinase-mediated autocrine loops (95, 96). Both pathways perform a critical function during development: Wnt/β-catenin signalling activates Mitf expression, and the Kit RTK is also absolutely required for the development of the neural crest-derived pigment cell population perhaps by controlling Mitf function/stability. Identification of key transcription factors (other than Mitf) that are regulated by these pathways will be crucial to dissecting the regulatory mechanisms that control melanoblast and melanoma proliferation.

Brn-2

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

In the melanocyte lineage the POU domain transcription factor Brn-2 (N-Oct3, POU3F2) first attracted attention as an unknown octamer-binding protein greatly over-expressed in melanoma cell lines (97). Subsequently this factor was identified as Brn-2 (98–100), that is also expressed in specific areas of the brain in most notably the hypothalamus and specific layers in the cortex and which is absolutely required for the development of the hypothalamus (101, 102). The elevated expression of Brn-2 observed in melanomas compared with melanocytes appears to arise through up-regulation of either of the two signalling pathways, Wnt/β-catenin or MAP kinase, that are deregulated in melanomas, suggesting that Brn-2 may function as positive regulator of melanoma survival/proliferation. Consistent with this, Brn-2 expression is substantially down-regulated by differentiating agents (103), and importantly Brn-2-specific siRNA-mediated gene silencing has confirmed the status of Brn-2 as a pro-proliferative transcription factor in melanoma (99, 104, 105). How Brn-2 acts to accelerate cell cycle progression is not known, but given the potential role of Mitf as a potential anti-proliferative factor, one attractive possibility is that Brn-2 will repress the Mitf promoter. Indeed, although Mitf expression may be observed in many melanomas as detected using an antibody recognizing all Mitf isoforms (106), Mitf-M expression was repressed in eight of 14 cell lines examined while re-expressing Mitf-M in an Mitf-negative melanoma cell line gave slower growth in vivo, although not in culture (79).

Intriguingly although both Brn-2 and Mitf are clearly targets for Wnt/β-catenin signalling only Mitf is expressed in neural crest-derived melanoblasts at embryonic day 11.5; Brn-2 expression in melanocytes occurs later in postnatal hair follicle melanocytes (105), and possibly earlier in the epidermis. Why Mitf and Brn-2 are activated by the same signalling pathways yet are differentially expressed is not known. Nevertheless Brn-2 is highly expressed in melanoblasts in culture where its expression is synergistically activated by fibroblast growth factor-2, stem cell factor, and endothelin-3 (107). Since these factors activate the MAP kinase signalling pathway it is likely that their effect on Brn-2 expression is similar to that observed in melanomas expressing activated BRAF. Presumably Brn-2 performs a pro-proliferative function in melanoblasts as it does in melanomas.

Conclusions – Multiple Roles for Mitf

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References

In summary, while the much of the necessary data are incomplete, the available evidence points to Mitf as a factor that exerts its effects at multiple levels. First, Mitf plays a key role in activating genes involved in differentiation functions such as melanogenesis. Second, Mitf regulates melanocyte morphology and migration through an ability to control the expression of factors involved in cytoskeletal organization and cell–cell adhesion. In this case much of the regulation may be indirect and mediated through factors such as Slug. Third, Mitf is likely to be a negative regulator of cell cycle progression and by preventing inappropriate cell division may prevent cells from undergoing apoptosis. In this respect the levels of active Mitf present in a cell must be tightly regulated, as indeed it is: too much and cells would not divide, too little and apoptosis might ensue. Fourth, the ability of Mitf to regulate transcription will be controlled by two opposing signalling pathways, namely the pro-proliferative MAP kinase pathway and the anti-proliferative stress-activated p38 pathway. In addition to Mitf, other key factors likely to play a role in melanocyte cell cycle control include the Mitf target Tbx2 and the Brn-2 transcription factor that have been characterized as playing an anti-senescence and pro-proliferative role, respectively. Taken together Mitf sits at the nexus of a complex series of regulatory networks that occupies the heart of melanocyte development and which also provides the key to understanding melanoma.

The overview presented here of Mitf as a multi-talented factor operating in the melanocyte lineage should be seen only as an impression that no doubt will change with time as more, and more reliable, information becomes available. Nevertheless, it is clear that Mitf resides at the heart of a transcription regulatory network that might be seen as a framework for the analysis of cell division, survival, migration and transformation in the fascinating system represented by the melanocyte lineage and melanoma.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Center of the Network – Microphthalmia-Associated Transcription Factor
  5. Mitf Expression
  6. Regulation of Mitf
  7. Mitf Targets and a Possible Role as a Negative Regulator of Proliferation
  8. Melanoma
  9. Brn-2
  10. Conclusions – Multiple Roles for Mitf
  11. References
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