No abstract

The clinically important melanoma diagnostic antibodies HMB-45, melan-A, and MITF (D5) recognize gene products of the melanocyte-lineage genes SILV/PMEL17/GP100, MLANA/MART1, and MITF, respectively. MITF encodes a transcription factor that is essential for normal melanocyte development and appears to regulate expression of several pigmentation genes. In this report, the possibility was examined that MITF might additionally regulate expression of the SILV and MLANA genes. Both genes contain conserved MITF consensus DNA sequences that were bound by MITF in vitro and in vivo, based on electrophoretic mobility shift assay and chromatin-immunoprecipitation. In addition, MITF regulated their promoter/enhancer regions in reporter assays, and up- or down-regulation of MITF produced corresponding modulation of endogenous SILV and MLANA in melanoma cells. Expression patterns were compared with these factors in a series of melanoma cell lines whose mutational status of the proto-oncogene BRAF was also known. SILV and MLANA expression correlated with MITF, while no clear correlation was seen relative to BRAF mutation. Finally, mRNA expression array analysis of primary human melanomas demonstrated a tight correlation in their expression levels in clinical tumor specimens. Collectively, this study links three important melanoma antigens into a common transcriptional pathway regulated by MITF.

The volumetric data rendered with both CBCT systems provided highly accurate data compared with the gold standard of physical measures directly from the skulls, with less than 1% relative error.

Determining the metastatic potential of intermediate thickness lesions remains a major challenge in the management of melanoma. Clinical studies have demonstrated that expression of melastatin/TRPM1 strongly predicts nonmetastatic propensity and correlates with improved outcome, leading to a national cooperative prospective study, which is ongoing currently. Similarly, the melanocytic markers MLANA/MART1 and MITF also have been shown to lose relative expression during melanoma progression. Recent studies have revealed that MITF, an essential transcription factor for melanocyte development, directly regulates expression of MLANA. This prompted examination of whether MITF also might transcriptionally regulate TRPM1 expression. The TRPM1 promoter contains multiple MITF consensus binding elements that were seen by chromatin immunoprecipitation to be occupied by endogenous MITF within melanoma cells. Endogenous TRPM1 expression responded strongly to MITF up-or down-regulation, as did TRPM1 promoter-driven reporters. In addition, MITF and TRPM1 mRNA levels were correlated tightly across a series of human melanoma cell lines. Mice homozygously mutated in MITF showed a dramatic decrease in TRPM1 expression. Finally, the slope of TRPM1 induction by MITF was particularly steep compared with other MITF target genes, suggesting it is a sensitive indicator of MITF expression and correspondingly of melanocytic differentiation. These studies identify MITF as a major transcriptional regulator of TRPM1 and suggest that its prognostic value may be linked to MITF-mediated regulation of cellular differentiation.

Malignant melanoma presents a substantial clinical challenge. Current diagnostic methods are limited in their ability to diagnose early disease and accurately predict individual risk of disease progression and outcome. The lack of adequate approaches to properly define disease subgroups precludes rational treatment design and selection. Better tools are urgently needed to provide more accurate

MITF and its related family members TFE3 and TFEB heterodimerize with each other, recognize the same DNA sequences, and are subject to many of the same post-translational modifications. We show that lysine residues within conserved small ubiquitin-like modifier (SUMO) consensus sites in these family members are subject to SUMO modification. Mutation of these sites significantly affects the transcriptional activity of MITF but does not alter dimerization, DNA binding, stability, or nuclear localization. Mutagenesis reducing the number of MITF binding sites in the promoter of TRPM1 from three to one eliminated the difference in transcriptional activity between the MITF mutants. Among other MITF target gene promoter constructs, differences in transcriptional activity between wild type and nonsumoylatable MITF were only seen in promoters with multiple MITF binding sites. These data support a synergy control model in which the functional consequences of MITF sumoylation depend on promoter context. Sumoylation, thus, provides a possible mechanism for altering the effects of MITF by affecting the target genes that it activates.MITF is a tissue-restricted, basic helix-loop-helix leucine zipper dimeric transcription factor. It is encoded by the mitf locus in mice (1) and when mutated leads to defects in melanocytes, the retinal pigment epithelium, mast cells, and osteosclasts. Mitf mutant mice are white due to a complete lack of melanocytes, whereas heterozygotes have a white belly spot (1, 2), demonstrating a requirement for mitf in production of this lineage. MITF continues to be necessary in the adult based on the existence of hypomorphic alleles in mice which cause postnatal melanocyte death and premature graying (3, 4). As a transcriptional mediator of differentiation, MITF acts downstream of the melanizing hormone ␣-melanocyte-stimulating hormone (5) and transcriptionally regulates the expression of the enzymes necessary for melanin production in differentiated melanocytes (for review, see Ref. 6). Although these data implicate MITF in both the survival and differentiation of melanocytes, little is known about biochemical regulatory pathways that control MITF in these different roles.MITF is part of the MiT transcription factor family whose members share significant homology and recognize the same DNA elements. Functionally, MITF binds to the canonical E-box promoter sequence CACGTG as well as to the nonpalindromic sequence CACATG (7,8). MITF functions as either a homodimer or as a heterodimer with the related MiT family transcription factors TFE3, TFEB, and TFEC (62). The related factor TFEB was recently identified as a translocated oncogene in papillary renal cell carcinoma in humans (10, 11). The structural features of these family members are so similar that MITF and TFE3 have been shown to genetically compensate for one another in regulation of osteoclast development in mice (12).Several post-translational modifications affect members of the MiT family. In melanocytes, activation of the mitogenactivated protei...

Waardenburg syndrome (WS) is an inherited sensorineural deafness condition in humans caused by melanocyte deficiencies in the inner ear and forelock. Mutation of microphthalmia-associated transcription factor (MITF) is known to produce WS type IIA whereas mutations of either endothelin (EDN) or its receptor endothelin receptor B (EDNRB) produce WS type IV. However, a link between MITF haploinsufficiency and EDN signaling has not yet been established. Here we demonstrate mechanistic connections between EDN and MITF and their functional importance in melanocytes. Addition of EDN to cultured human melanocytes stimulated the phosphorylation of MITF in an EDNRB-dependent manner, which was completely abolished by mitogen-activated protein kinase kinase inhibition. The expression of melanocyte-specific MITF mRNA transcripts was markedly augmented after incubation with EDN1 and was followed by increased expression of MITF protein. Up-regulated expression of MITF was found to be mediated via both the mitogen-activated protein kinase-p90 ribosomal S6 kinase-cAMP response element-binding protein (CREB) and cAMP-protein kinase A-CREB pathways. In addition, EDNRB expression itself was seen to be dependent on MITF. The functional importance of these connections is illustrated by the ability of EDN to stimulate expression of melanocytic pigmentation and proliferation markers in an MITF-dependent fashion. Collectively these data provide mechanistic and epistatic links between MITF and EDN/EDNRB, critical melanocytic survival factors and WS genes.

Computational simulations which include three-dimensional (3-D) image processing and biomechanical calculations should provide useful information to our research and orthodontic clinic as a clinical tool defined as 'thinking'. In this review, 1) biomechanical simulations applied to predict the mandibular growth; 2) mathematical models of virtual bone cells and 3) 3-D images and solid model simulations for surgical planning are introduced. In biomechanical simulation, biting force, electromyographic (EMG) activity and cephalograms of 32 subjects were applied. Computational results of mathematical model were compared with actual bone growth in a rat. Three-dimensional image and solid model of 14 patients were utilized for their treatment planning. From the results, several concepts of our simulations were confirmed: 1) reaction forces generated by masticatory muscles at the condyle control the direction of mandibular growth; 2) some mathematical models have the possibility to describe the process of bone growth; 3) 3-D image processing software and solid models are necessary for diagnosis and planning of orthognathic surgery. We also believe that the orthodontists can more accurately predict the affects of surgical procedures and orthodontic tooth movement using the new cone beam X-ray computed tomography (CT) (CB MercuRay; Hitachi Medico Technology, Tokyo, Japan) and its advanced application software.