Function Of Microphthalmia-associated Transcription Factor Research Articles

The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300

The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma.

Microphthalmia-Associated Transcription Factor-Dependent Melanoma Cell Adhesion Molecule Activation Promotes Peritoneal Metastasis of Ovarian Cancer.

Ovarian cancer (OvCa) is one of the leading causes of death due to its high metastasis rate to the peritoneum. Recurrent peritoneal tumors also develop despite the use of conventional platinum-based chemotherapies. Therefore, it is still important to explore the factors associated with peritoneal metastasis, as these predict the prognosis of patients with OvCa. In this study, we investigated the function of microphthalmia-associated transcription factor (MITF), which contributes to the development of melanoma, in epithelial ovarian cancer (OvCa). High MITF expression was significantly associated with a poor prognosis in OvCa. Notably, MITF contributed to the motility and invasion of OvCa cells, and specifically with their peri-mesothelial migration. In addition, MITF-positive cells expressed the melanoma cell adhesion molecule (MCAM/CD146), which was initially identified as a marker of melanoma progression and metastasis, and MCAM expression was regulated by MITF. MCAM was also identified as a significant prognostic factor for poor progression-free survival in patients with OvCa. Collectively, our results suggest that MITF is a novel therapeutic target that potentially promotes peritoneal metastasis of OvCa.

Structural and Functional Insights into TAZ2‐mediated CBP/p300 Recruitment by the Melanogenic Transcription Factor MITF

The microphthalmia‐associated transcription factor (MITF) is a master regulator of development and differentiation within the melanocyte lineage. However, aberrant MITF activity can lead to multiple malignancies such as skin cancer, where it plays a key role in modulating the proliferation and invasiveness of melanoma. MITF is a basic helix‐loop‐helix leucine zipper transcription factor that binds to specific gene promoters via a central DNA‐binding domain. MITF also recruits transcriptional co‐activators, such as the histone acetyltransferase CREB‐binding protein and its homologue p300 (CBP/p300) through an N‐terminal acidic transactivation domain (TAD), however the details of these interactions are not yet fully understood. In order to gain insight into the mechanisms of gene regulation by MITF, we investigated the structure and functional interaction between MITF‐TAD and the transcription adapter putative zinc finger (TAZ2) domain of CBP/p300. In mammalian‐one‐hybrid assays MITF transcriptional activity was enhanced in the presence of co‐transfected CBP/p300 and abolished upon deletion of residues within the MITF‐TAD. Peptide microarrays indicated that no one residue of MITF is essential for TAZ2 binding, however, deletion of multiple residues in MITF‐TAD ablated its ability to bind TAZ2. NMR‐based chemical shift mapping experiments determined that MITF‐TAD interacts with the same surface of TAZ2 as the adenoviral protein E1A, which has been shown to inhibit MITF function. We determined that E1A and MITF‐TAD directly compete for CBP/p300 through the TAZ2 domain using NMR‐based titrations, pulldown, and mammalian‐hybrid assays. Furthermore, we used qPCR to measure the effect of disrupting these interactions on the transcription of MITF‐specific target genes in melanoma‐derived cell lines. Understanding mechanistic details regarding the interaction between MITF and its co‐activators is fundamental to our understanding of gene regulation by MITF and may outline a potential new strategy to inhibit MITF function.Support or Funding InformationThis research was funded by the Beatrice Hunter Cancer Research Institute, Nova Scotia Heath Research Foundation Scotia Scholars Award, a Canadian Institutes of Health Research studentship, Dalhousie Nova Scotia Graduate Scholarship, and Izaak Walton Killam Predoctoral Scholarship.

MITF-the first 25 years.

All transcription factors are equal, but some are more equal than others. In the 25 yr since the gene encoding the microphthalmia-associated transcription factor (MITF) was first isolated, MITF has emerged as a key coordinator of many aspects of melanocyte and melanoma biology. Like all transcription factors, MITF binds to specific DNA sequences and up-regulates or down-regulates its target genes. What marks MITF as being remarkable among its peers is the sheer range of biological processes that it appears to coordinate. These include cell survival, differentiation, proliferation, invasion, senescence, metabolism, and DNA damage repair. In this article we present our current understanding of MITF's role and regulation in development and disease, as well as those of the MITF-related factors TFEB and TFE3, and highlight key areas where our knowledge of MITF regulation and function is limited.

Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter

To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR) promoter. Methods: The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, respectively. Results: IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted. Conclusion: IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.

Microphthalmia-associated transcription factor in melanoma development and MAP-kinase pathway targeted therapy.

SummaryMalignant melanoma is a neoplasm of melanocytes, and the microphthalmia‐associated transcription factor (MITF) is essential for the existence of melanocytes. MITF's relevance for this cell lineage is maintained in melanoma, where it is an important regulator of survival and balances melanoma cell proliferation with terminal differentiation (pigmentation). The MITF gene is amplified in ~20% of melanomas and MITF mutation can predispose to melanoma development. Furthermore, the regulation of MITF expression and function is strongly linked to the BRAF/MEK/ERK/MAP‐kinase (MAPK) pathway, which is deregulated in >90% of melanomas and central target of current therapies. MITF expression in melanoma is heterogeneous, and recent findings highlight the relevance of this heterogeneity for the response of melanoma to MAPK pathway targeting drugs, as well as for MITF's role in melanoma progression. This review aims to provide an updated overview on the regulation of MITF function and plasticity in melanoma with a focus on its link to MAPK signaling.

MITF in melanoma: mechanisms behind its expression and activity.

MITF (microphthalmia-associated transcription factor) represents a melanocytic lineage-specific transcription factor whose role is profoundly extended in malignant melanoma. Over the last few years, the function of MITF has been tightly connected to plasticity of melanoma cells. MITF participates in executing diverse melanoma phenotypes defined by distinct gene expression profiles. Mutation-dependent alterations in MITF expression and activity have been found in a relatively small subset of melanomas. MITF activity is rather modulated by its upstream activators and suppressors operating on transcriptional, post-transcriptional and post-translational levels. These regulatory mechanisms also include epigenetic and microenvironmental signals. Several transcription factors and signaling pathways involved in the regulation of MITF expression and/or activity such as the Wnt/β-catenin pathway are broadly utilized by various types of tumors, whereas others, e.g., BRAFV600E/ERK1/2 are more specific for melanoma. Furthermore, the MITF activity can be affected by the availability of transcriptional co-partners that are often redirected by MITF from their own canonical signaling pathways. In this review, we discuss the complexity of a multilevel regulation of MITF expression and activity that underlies distinct context-related phenotypes of melanoma and might explain diverse responses of melanoma patients to currently used therapeutics.

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Caffeic Acid Phenethyl Ester Inhibits Alpha-Melanocyte Stimulating Hormone-Induced Melanin Synthesis through Suppressing Transactivation Activity of Microphthalmia-Associated Transcription Factor

Caffeic acid phenethyl ester (1), a natural compound found in various plants and propolis, is a well-known anti-inflammatory, immunomodulatory, and cytotoxic agent. The present study aimed to investigate the molecular events underlying the antimelanogenic activity of 1 in alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16-F10 melanoma cells. In this investigation, 1 effectively reduced α-MSH-stimulated melanin synthesis by suppressing expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2), although this compound did not directly inhibit tyrosinase enzyme activity. On the other hand, the expression and nuclear translocation of microphthalmia-associated transcription factor (MITF) as a key transcription factor for tyrosinase expression regulating melanogenesis were not affected by treatment with 1. The upstream signaling pathways including cAMP response element-binding protein (CREB), glycogen synthase kinase-3β (GSK-3β), and Akt for activation and expression of MITF were also not influenced by 1. Interestingly, 1 inhibited transcriptional activity of a tyrosinase promoter by suppressing the interaction of MITF protein with an M-box containing a CATGTG motif on the tyrosinase promoter. Given the important role of MITF in melanogenesis, suppression of 1 on the function of MITF to transactivate tyrosinase promoter may present a novel therapeutic approach to treat hyperpigmentation disorders.

BRG1 promotes survival of UV‐irradiated melanoma cells by cooperating with MITF to activate the melanoma inhibitor of apoptosis gene

Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.

Frequent mutations in the MITF pathway in melanoma

Microphthalmia-associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient-matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix-loop-helix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia-associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

Myocyte Enhancer Factor 2 and Microphthalmia-associated Transcription Factor Cooperate with NFATc1 to Transactivate the V-ATPase d2 Promoter during RANKL-induced Osteoclastogenesis

The V-ATPase d2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF overexpression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis.

MiTF Regulates Cellular Response to Reactive Oxygen Species through Transcriptional Regulation of APE-1/Ref-1

Microphthalmia-associated transcription factor (MiTF) is a key transcription factor for melanocyte lineage survival. Most previous work on this gene has been focused on its role in development. A role in carcinogenesis has emerged recently, but the mechanism is unclear. We classified melanoma cells into MiTF-positive and -negative groups and explored the function of MiTF in regulating cellular responses to reactive oxygen species (ROS). The MiTF-positive melanoma cell lines accumulated high levels of apurinic/apyrimidinic endonuclease (APE-1/Ref-1, redox effector-1), a key redox sensor and DNA endonuclease critical for oxidative DNA damage repair. We demonstrate that APE-1 is a transcriptional target for MiTF. Knocking down MiTF led to reduced APE-1 protein accumulation, as well as abolished induction of APE-1 by ROS. MiTF-negative melanoma cells survived more poorly under ROS stress than the MiTF-positive cells based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Trypan blue staining. Overexpression of APE-1 partially rescued ROS-induced cell death when MiTF was depleted. We conclude that MiTF regulates cellular response to ROS by regulation of APE-1, and this may provide a mechanism of how MiTF is involved in melanoma carcinogenesis.

Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes

Upregulation of microphthalmia-associated transcription factor (MITF) expression has been proposed to mediate melanogenesis stimulated by cAMP, whereas downregulation of MITF has been suggested to underlie the depigmentary effects of resveratrol, a promising chemotherapeutic found in red wine. We have assessed the contribution of MITF to pigmentation regulation by treating primary cultures of normal human melanocytes with the adenylate cyclase activator forskolin and/or resveratrol, then quantifying mRNA and protein levels for MITF, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase (DCT). The inhibition of tyrosinase activity by resveratrol was not due to alterations in MITF, but instead was explained by both direct tyrosinase inhibition and a post-transcriptional effect that reduced the amount of fully processed tyrosinase. Glycosidase digestion revealed that the basis for the tyrosinase decrease was the retention of an immature form in the ER and subsequent loss of the mature, Golgi-processed enzyme. Elevation of intracellular cAMP by forskolin markedly increased protein levels for MITF, tyrosinase and DCT, however there was no concomitant increase in tyrosinase or DCT mRNA. This indicated that elevated levels of MITF were not sufficient to promote transcription of these melanogenic genes and that the increase in their protein abundance appeared to be predominantly mediated through post-transcriptional processing events.

Suppression of Progressive Loss of Coat Color in Microphthalmia-Vitiligo Mutant Mice

The coat color of C57BL/6-Mitfvit/vit mice whitens with age, because of a one-nucleotide mutation in the DNA-binding region of the microphthalmia-associated transcription factor (MITF), which plays an important role in melanocyte growth and differentiation. To investigate the signals regulating MITF function, we prepared transgenic mice expressing three of the external signals that are important for melanocyte development, i.e., hepatocyte growth factor (HGF), stem cell factor (SCF), and endothelin-3 (ET3), and crossed these mice with Mitfvit/vit mice. We found that the age-dependent coat color whitening of the Mitfvit/vit mice was completely suppressed by the overexpression of HGF or SCF in the skin, but not by that of ET3. Moreover, HGF, but not ET3, promoted the proliferation of Mitfvit/vit mice-derived melanocytes in culture. These results suggest that the signals from exogenous HGF and SCF rescued the mi-vitiligo mutation and also that ET3 does not stimulate the common signal transduction pathway for MITF activation shared by HGF and SCF.

Glycogen Synthase Kinase 3β Is Activated by cAMP and Plays an Active Role in the Regulation of Melanogenesis

In human and mouse, cAMP plays a key role in the control of pigmentation. cAMP, through the activation of protein kinase A, increases the expression of microphthalmia-associated transcription factor (MITF), which in turn stimulates tyrosinase gene expression, to allow melanin synthesis. Beyond this simplified scheme, cAMP inhibits phosphatidylinositol 3-kinase (PI3K), and inhibition of PI3K, by a specific inhibitor, stimulates melanogenesis. However, the link between the PI3K pathway and melanogenesis remained to be elucidated. In this report, we showed that cAMP, through a protein kinase A-independent mechanism, led to inhibition of AKT phosphorylation and activity. Consistent with the role of AKT in the regulation of glycogen synthase kinase 3beta (GSK3beta), cAMP decreased the phosphorylation of GSK3beta and stimulated its activity. Further, experiments were performed to investigate the role of GSK3beta in the regulation of MITF expression and function. We observed that GSK3beta regulated neither MITF promoter activity nor the intrinsic transcriptional activity of MITF but synergized with MITF to activate the tyrosinase promoter. Additionally, lithium, a GSK3beta inhibitor, impaired the response of the tyrosinase promoter to cAMP, and cAMP increased the binding of MITF to the M-box. Taking into account that GSK3beta phosphorylates MITF and increases the ability of MITF to bind its target sequence, our results indicate that activation of GSK3beta by cAMP facilitates MITF binding to the tyrosinase promoter, thereby leading to stimulation of melanogenesis.

Regulation of the Microphthalmia-associated Transcription Factor Gene by the Waardenburg Syndrome Type 4 Gene,SOX10

The absence of melanocytes from the cochlea and epidermis is responsible of deafness and hypopigmentation, two symptoms shared by the four Waardenburg syndrome (WS) subtypes. Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation. Mutations, which impair MITF function or expression, result in an abnormal melanocyte development leading to the WS2. WS1 and WS3 are caused by mutation in the gene encoding the transcription factor Pax3, which regulates MITF expression. Recently, mutations in SOX10, a gene encoding a SRY-related transcription factor, have been reported in patients with WS4. However, the molecular basis of the defective melanocyte development in these patients remained to be elucidated. In the present report, we demonstrate that Sox10 is a strong activator of the MITF promoter, and we identify a Sox10 binding site between -264 and -266 of the MITF promoter. Finally, we show that three SOX10 mutations found in WS4 abolish the transcriptional activity of the resulting Sox10 proteins toward the MITF promoter. Taken together, our observations bring new and meaningful information concerning the molecular process that leads to a defective melanocyte development in WS4 patients with SOX10 mutations.

Cyclic AMP a key messenger in the regulation of skin pigmentation.

Compelling evidence has been gathered indicating that pro-opiomelanocortin peptides, alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH), through the cyclic AMP pathway, play a pivotal role in melanocyte differentiation and in the regulation of melanogenesis. Recently, the molecular events linking cAMP to melanogenesis up-regulation have been elucidated. This cascade involves the activation of protein kinase A and CREB transcription factor, leading to the up-regulation of the expression of Microphthalmia associated transcription factor (MITF). MITF has been found mutated in patients with Waardenburg syndrome 2A, and plays a crucial role in melanocyte development. MITF binds and activates melanogenic gene promoters, thereby increasing their expression which results in an increased melanin synthesis. Beyond this simplified scheme, It appears that melanogenic gene expression is controlled by a complex network of regulation involving other transcription factors such as Brn2, TBX2, PAX3 and SOX10. Further studies are required to better understand the respective roles of these factors in the regulation of melanin synthesis. In addition, other intracellular signaling pathways, like the phosphatidyl inositol 3-kinase pathway, as well as the molecular cascade of events governed by the small GTP-binding protein Rho, seem to be involved in the regulation of melanogenesis and melanocyte dendricity. Finally, it should be mentioned that cAMP activates a melanocyte-specific pathway leading to MAP kinase activation. MAP kinase, ERK2, phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. Thus, in addition to the complex transcriptional regulation, melanogenesis is also subjected to a post-translational regulation that controls MITF or tyrosinase function. Taken together, these complex molecular processes would finally allow a fine tuning of melanocyte differentiation leading to melanin synthesis.

Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance

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

Characterization of Density-Dependent Regulation of the Tyrosinase Gene Promoter: Role of Protein Kinase C

The rate-limiting step in melanogenesis is catalyzed by tyrosinase, a multifunctional enzyme encoded by the albinolocus. We have previously reported that depletion of protein kinase C by long-term treatment of B16 mouse melanoma cells with phorbol dibutyrate (PDBu) prevented cell density-dependent melanogenesis. This was accompanied by a lack of induction of tyrosinase protein and mRNA. We report here the effect of PDBu on the functional activity of the mouse tyrosinase promoter by reporter gene assay and its effect on the binding of nuclear proteins from B16 cells to the “M-box” region of the mouse tyrosinase promoter. Short-term PDBu treatment of B16 cells transfected with a mouse tyrosinase promoter–luciferase construct resulted in increased reporter gene activity, while long-term PDBu treatment inhibited reporter gene activity. Using an oligonucleotide containing the M-box and its flanking residues in electrophoretic mobility shift assays, we found a density-dependent change in the pattern of DNA–protein complexes. One complex was found to be negatively regulated by long-term PDBu treatment. Competition experiments with various mutated oligonucleotides demonstrated that both the M-box and flanking residues are important for nuclear protein binding. The complex whose formation was inhibited by long-term PDBu treatment was shown to contain the basic helix-loop-helix leucine zipper protein microphthalmia-associated transcription factor (MITF). These results suggest that chronic PDBu treatment might inhibit tyrosinase expression (and subsequent melanogenesis) by affecting the amount or function of MITF.