Gene Expression Regulation by Agonist-Independent Constitutive Signaling of Melanocortin-1 Receptor

BackgroundMelanocortin-1 receptor (Mc1r), a key signaling receptor for melanogenesis, has been reported to mediate migration of B16F10 melanoma cells. Interestingly, this activity appears to be a part of the constitutive signaling of Mc1r.MethodsWe carried out small interfering RNA-mediated knock-down of Mc1r on murine melanoma B16F10 cells and performed microarray analysis to characterize changes in the gene expression profile.ResultsWe isolated 22 and four genes whose expression decreased and increased, respectively, by 2.5-fold or higher as the result of Mc1r knock-down. Several down-regulated genes have been proposed to be involved in cell migration. Among these genes are several members of the chemokine gene family.ConclusionWe provide a gene set for further functional analyses of Mc1r. The Mc1r target genes we present may be particularly relevant for understanding the ligand-independent activity of Mc1r. Further examination of the mode of action may lead to novel strategies in regulating the migration and metastasis of melanoma cells

Identification of direct regulatory targets of the transcription factor Sox10 based on function and conservation

Abstract Background Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs. Results We describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin) were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay. Conclusion In sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.</p

Mc1r promotes migration of melanoma cells.

<p>(A) Quantitative Real time RTPCR assays were carried out using B16F10 murine melanoma cells transfected with the universal control siRNA, MT1-Mc1r, or one of the three siRNAs specific for Mc1r, WT1-Mc1r, WT2-Mc1r, and WT3-Mc1r. The nucleotide sequence of MT1-Mc1r differs from that of WT1-Mc1r by 5 nucleotides. The expression levels of Mc1r, Ald1a, and Ctbp1 were examined. The effect of Mc1r knockdown is expressed relative to that of the universal control siRNA after normalization with GAPDH expression level. Values represent the average of three independent real-time PCR experiments each carried out in duplicates, and error bars represent standard deviations. (B–G) B16F10 cells were treated with the universal control siRNA (B), MT1-Mc1r (C), WT1-Mc1r (D), WT2-Mc1r (E), or WT3-Mc1r (F) and put to transwell migration assay. (G) Quantitation of transwell migration assay. The effect of Mc1r knockdown on the number of cells that migrated through the filter pores is shown in percentile relative to the universal control case. Values represent the average of 5 independent trials, and error bars represent standard deviations. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p

Knockdown of Sox10 leads to inhibition of migration of B16F10 melanoma cells.

<p>B16F10 murine melanoma cells were transfected with control siRNAs, MT1-Sox10 (A, B; MT, mutant) and MT2-Sox10 (E, F) or siRNAs specific for Sox10, WT1-Sox10 (C, D; WT, wild type) and WT2-Sox10 (G, H). Nuclei were stained with DAPI (A, C, E, G) and anti-Sox10 antibody (B, D, F, H). Nucleotide sequences of MT1-Sox10 and MT2-Sox10 differ from those of WT1-Sox10 and WT2-Sox10 by 5 nucleotides respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031477#pone.0031477.s007" target="_blank">Table S1</a>). Sox10 was down-regulated only with WT siRNAs (D, H) but not with MT siRNAs (B, F). B16F10 cells treated with siRNAs were put to transwell migration assay (I–M). Transfection of WT1-Sox10 (K) and WT2-siRNA (L) led to significant reduction in migration of the cells compared to transfection of MT1-Sox10 (I) or MT2-Sox10 (J). (M) Quantitation of transwell migration assay. The effect of Sox10 knockdown on the number of cells that migrated through the filter pores is shown in percentile relative to the matching control case. Values represent the average of 5 independent trials, and error bars represent standard deviations. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p

Confirmation of microarray expression profiling.

<p>(A) Quantitative real time RTPCR assays were carried out using B16F10 cells transfected with the MT1-Sox10 or WT1-Sox10. A subset of genes that showed down-regulation by WT1-Sox10 in the microarray assay by 2.5 fold or higher in all triplicates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031477#pone-0031477-t001" target="_blank">Table 1</a>) and two non-target genes whose expression levels were unchanged (Ald1a and Ctbp1) were used to validate the results from the microarray assay. The effect of Sox10 knockdown by the specific siRNA on the expression level of each target gene is expressed relative to that of the control siRNA after normalization with GAPDH expression level. Values represent the average of three independent real-time PCR experiments each carried out in duplicates, and error bars represent standard deviations. (B) Real time RTPCR carried out with MT2-Sox10 and WT2-Sox10. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p

List of genes down-regulated by WT1-Sox10.

<p>Fold change is in comparison to MT1-Sox10 transfected cells. Microarray screening was carried out in triplicates. Genes that show down-regulation by 2.5 fold or higher in all triplicates are listed. Oculospanin (oscp) is a synonym for tetraspanin 10 (Tspan10).</p

Confirmation of the role of Mc1r on cell migration using an in vivo metastasis model.

<p>(A, B) Effect of Mc1r knockdown on the development of pulmonary metastatic colony was determined. B16F10 melanoma cells were treated with MT1-Mc1r (A) or WT1-Mc1r (B) and injected into tail vein of C57BL/6 mice. Representative lungs harvested after 18 days are shown. (C) B16F10 colonies visible on the lung surface were counted and plotted. N = 9 for MT1-Mc1r and N = 8 for WT1-Mc1r. The significance of difference (p<0.0001) was determined by <i>t</i>-test.</p

Targets of Sox10 regulate migration of B16F10 melanoma cells.

<p>(A) Quantitative RTPCR assay was carried out to test the efficacy of siRNAs directed for the selected genes. For each gene, the knockdown effect of the specific siRNA is expressed relative to that of a universal control siRNA (siCONTROL Non-Targeting siRNA #2) after normalization with GAPDH expression level. Values represent the average of three independent real-time PCR experiments each carried out in duplicates, and error bars represent standard deviations. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05. (B-I) B16F10 cells were treated with the universal control siRNA, WT1-Sox10 or a specific siRNA for each of the selected Sox10 target genes and subjected to transwell migration assay: Control (B), Sox10 (C), Hyal 1 (D), Lims2 (E), Mc1r (F), P2ry2 (G), Tm7sf1 (H), and Tspan10 (I). Representative results are presented.</p

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells*

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, α-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by α-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells