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• W1953331147 abstract "The importance of melanoblast migration is underscored by the fact that its disruption, for instance by genetic mutations, can lead to developmental pigmentation defects of the skin (1, S1–S3). Melanoblast migration, however, is not limited to development (S4–S5). In the adult, melanoblasts also migrate during skin wound healing and during repigmentation of vitiligo lesions. In these processes, melanoblasts migrate from hair follicles into the wounded or depigmented areas 2, 3. Furthermore, upon transformation, melanocytes can re-initiate migration during the formation of melanomas and their metastases 4. Melanocyte migration is regulated by numerous genes including β-catenin and Mitf (5, S6–S10). The encoded protein, MITF, is a basic-helix-loop-helix-leucine zipper (bHLHZip) transcription factor (S11). It usually acts as a transcriptional activator that binds canonical E-box promoter sequences including CATGTG and CACGTG (S12) and regulates a variety of target genes (S13), which in turn are involved in cell cycle regulation (S14), cell survival (S15–S16), cell differentiation (S17–S19) and cell migration 5. The role of MITF in this latter process, however, has attracted relatively little attention. Here, we approach the question of the role of MITF in melanoblast migration by experimentally manipulating the levels of MITF in a melanoblast cell line, melb-a. Our results show that MITF is capable of directly suppressing the expression of Mcam (melanoma cell adhesion molecule) and that increased expression of Mcam after Mitf knockdown promotes migration of melb-a cells while concomitant knockdown of both Mitf and Mcam again reduces cell migration. Hence, it appears that MITF and MCAM are involved in a direct pathway regulating melanoblast migration. All experiments were conducted on melanoblast cells using methods (for details, see Data S1). We first tested the role of MITF in melb-a melanoblast migration by wound healing and a transwell migration assay. Twenty-four hours after a transfected monolayer was wounded, melb-a cells transfected with siRNAs specific for Mitf (si-Mitf-1 or si-Mitf-2) migrated into the wound area. Control siRNA (si-C)-transfected cells also migrated, but by comparison, their migration was much slower (Figure S1A,B, Fig. 2b). In transwell assays, si-Mitf-1 or si-Mitf-2 transfection increased the number of cells that crossed the membrane by twofold compared to si-C-transfected cells (Figure S1C,D, Fig. 2c,d). Moreover, overexpression of MITF reduced cell migration compared to overexpression of GFP (Figure S1e–g). These results suggest that low levels of MITF promote, and high levels inhibit, melanoblast migration. Next, we tested whether Mitf knockdown would lead to changes in the expression levels of a number of genes previously implicated in regulating cell migration (Table S1). Indeed, the expression of some genes, including several cadherin genes, was also downregulated while that of others was upregulated (Fig. 1a). In particular, the expression of one gene, Mcam (also known as CD146,MUC18 and METMCAM), was significantly increased after Mitf knockdown (Fig 1b). In contrast, overexpression of Mitf led to a decrease in Mcam expression (Fig 1c). MCAM is of interest because it has previously been shown to be involved in melanoma metastasis 6. To test whether MITF represses MCAM by directly binding Mcam regulatory sequences, we employed standard chromatin-IP and promoter mutation analyses. In fact, the Mcam gene contains a conserved MITF binding E-box element (TCACTTG) just 2-bp downstream of the Mcam transcriptional start site (Fig. 1d). Standard chromatin-IP showed amplification of a corresponding amplicon after MITF immunoprecipitation but not after control IgG immunoprecipitation (Fig. 1e). Luciferase reporter assays, performed in Hela cells, showed reduced activity of a corresponding Mcam promoter fragment after Mitf cotransfection compared to GFP control cotransfection (Fig 1f). Deletion of the E-box, or its mutation, clearly interfered with this MITF-mediated downregulation of promoter activity (Fig. 1f). These results suggest that MITF directly binds to the Mcam promoter and represses its transcriptional activity. To elucidate the functional roles of MCAM in melanoblast migration, we either reduced MCAM expression in melb-a cells by si-Mcam-1 or si-Mcam-2 or overexpressed it using a lentivirus system. As shown in Figures S2 and S3, knockdown of Mcam inhibits melanocyte migration and overexpression promotes it. The above experiments indicated that downregulation of Mitf promotes cell migration by increasing Mcam expression. Hence, simultaneous downregulation of Mitf and Mcam should correct the effect of selective Mitf downregulation. As shown in Fig. 2a,b, this was indeed the case as si-Mitf singly transfected cells, or cells doubly transfected with si-Mitf and a control siRNA, filled in a wounded area more readily than si-Mitf/si-Mcam doubly transfected cells. These results, confirmed in corresponding transwell assays (Fig. 2c,d), suggest that MITF and MCAM are linked in a direct linear pathway to affect cell migratory behaviours. Here, we show that MITF inhibits melanoblast migration as a transcriptional repressor of a cell adhesion molecule, MCAM, which has originally been identified as a marker that distinguishes benign nevi from melanomas whose metastasis it promotes 6. MCAM is detected in several melanocytic cell lines (7, S20–S21), although its regulation and function are largely unexplored. Given the multiple target genes MITF regulates in melanocytes, it is not surprising that MITF is also involved in the regulation of MCAM. Nevertheless, most MITF target genes are stimulated by MITF, even though some, such as ZEB1 and SHC4, are apparently suppressed 8, 9. Hence, our findings add MCAM to the list of MITF target genes that are negatively regulated by this multifunctional transcription factor. In sum, our results provide new evidence that MITF works as a transcriptional repressor to play a functional role in regulating melanoblast migration. This finding may have implications for our understanding of skin wound healing, melanocyte repopulation in vitiligo lesions and melanoma invasion and metastasis. We thank for Drs. Dorothy Bennett, Ming Zhang and Zhihai Qin for reagents and Dr. Heinz Arnheiter for reagents and thoughtful comments on the manuscript. This work was supported by NSFC (30971467, 31171408), Zhejiang Provincial NSF (LZ12C12001, Q14C120004, LQ13H120004). BR/SZ designed/performed experiments and analysed data. XM/XZ/YL performed experiments. FL/JQ contributed reagents. LH designed experiments, analysed data and wrote the manuscript. The authors state no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. *For more detailed information see supporting literature references provided in Data S2." @default.
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• W1953331147 date "2015-09-15" @default.
• W1953331147 modified "2023-10-14" @default.
• W1953331147 title "Microphthalmia-associated transcription factor regulates skin melanoblast migration by repressing the melanoma cell adhesion molecule" @default.
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• W1953331147 doi "https://doi.org/10.1111/exd.12835" @default.
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