β-Catenin Promotes the Differentiation of Epidermal Langerhans Dendritic Cells

The epithelial signaling protein and transcriptional regulator β-catenin has recently been implicated in hematopoietic dendritic cell (DC) differentiation as well as in DC-mediated tolerance. We here observed that epidermal Langerhans cells (LCs) but not interstitial/dermal DCs express detectable β-catenin. LCs are unique among the DC family members in that LC networks critically depend on epithelial adhesion molecules as well as on the cytokine transforming growth factor-β1 (TGF-β1). However, despite the important functions of LCs in the immune system, the molecular mechanisms governing LC differentiation and maintenance remain poorly defined. We found that TGF-β1 induces β-catenin in progenitor cells undergoing LC differentiation and that β-catenin promotes LC differentiation. Vitamin D, another epidermal signal, enhanced TGF-β1-mediated β-catenin induction and promoted the expression of multiple epithelial genes by LCs. Moreover, full-length vitamin D receptor (VDR) promoted, whereas a truncated VDR diminished, the positive effects of ectopic β-catenin on LC differentiation. Therefore, we here identified β-catenin as a positive regulator of LC differentiation in response to TGF-β1 and identified a functional interaction between β-catenin and VDR in these cells

A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter

A CpG island (CGI) lies at the 5′ end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

<div><p>Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.</p></div

α-Catulin promotes NF-κB activation in human primary melanocytes and melanoma cells.

<p>(<b>A</b>) A 5x NF-B-luc reporter gene (0.25μg) was co-transfected into melanocytes together with different concentrations of α-catulin (1 or 1.5 μg) with or without IKK-β(0.5μg) or p65 (0.5μg). 24 h later cells were non-stimulated or stimulated with TNF-α or LPS. Luciferase levels were normalized for a co-transfected RFP control (0.25μg). (<b>B</b>) Mel.7, Mel.15 and Mel.17 cells were stable infected with lentiviral particles containing a vector-based mirRNA construct directed against α-catulin (sh-catu 1 or sh-catu2; Materials and Methods), and α-catulin mRNA levels were analyzed by real-time PCR. (<b>C</b>) Mel.7, Mel.15 and Mel.17 cells were analyzed by Western blot with antibodies against α-catulin. (<b>D</b>) A NF-κB-luc reporter gene was tranfected into melanocytes and different melanoma cells containing stable integrated α-catulin mirRNA constructs (α-catulin-knockdown), and luciferase values were determined 24 h later and normalized for co-transfected JRED values. (<b>E</b>) Melanoma 7 cells as in (<b>D</b>) except that the cells were stimulated with TNFα, LPS, HGF and 10% FCS for further 8 h. (<b>F</b>) Mel.7 cells (n.s., sh-catu1/2) were transfected with NF-κB reporter plasmid and with or without si-RNA construct directed against E-cadherin and luciferase values were determined. (<b>G</b>) NF-κB-luc reporter assay with A375 melanoma cells transfected with mock, myc-α-catulin or sh-catu2 plasmids together with or without E-cadherin si-RNA. *Indicates P>0.005, **P>0.001, ***P>0.0001, Student´s <i>t</i> test.</p

α-Catulin knockdown reduces phosphorylation of ERK, JNK and c-Jun in cisplatin treated melanoma cells.

<p>Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0, 5, 10 and 20 μg/ml cisplatin for 24h and analyzed by Western blot with antibodies against (<b>A</b>) p-ERK, total ERK, (<b>B</b>) p-JNK, total JNK, (<b>C</b>) p-c-Jun (<b>D</b>) Mcl-1 and CBP. GAPDH or Tubulin were used as loading control.</p

α-Catulin knockdown enhances susceptibility of melanoma cells to cisplatin.

<p>(<b>A-F</b>) Stable infected (n.s., sh-catu2) (<b>A</b>) Mel.7, (<b>B</b>) Mel.17, (<b>C</b>) Mel.15, (<b>D</b>) Mel.7 spheroids, (<b>E</b>) Mel.7 (n.s., sh-catu1) or (<b>F</b>) Melanocytes (mock, myc-α-catulin) were treated with different concentrations of cisplatin for 48h and cell survival normalized to untreated cells (pos. contr.). Viability was analyzed by CellTiter-Blue Assay. (<b>G</b>) Stable infected Mel.7 spheroids were treated with 200 μg/ml cisplatin for 96 hours and diameter of the spheroids determined before and after treatment and statistically evaluated. Observations (•) mean (-) n = 14, (<b>H</b>) Microscopic images from Mel.7 spheroids.</p

α-Catulin knockdown enhances apoptosis in cisplatin-treated melanoma cells.

<p>(<b>A</b>) Stable infected Mel.7 cells (n.s., sh-catu1, sh-catu2) were treated with 0 and 10 μg/ml cisplatin for 48 h and stained with Annexin V and PI and analysed using flow cytometry (<b>B</b>) Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0 and 2.5μg/ml cisplatin for 6 h. For cytochrome c release assay, cells were treated with permeabilization buffer, fixed with formaldehyde, stained with antibody against cytochrome c and analyzed by Flow Cytometry. (<b>C</b>) Mel.7 cells (n.s., sh-catu2) were seeded in a 96 well plate and treated with 0, 2.5, 5, 10 or 20 μg/ml cisplatin for 6 h and mitochondrial membrane potential was determined using JC-1 assay (<b>D</b>) Mel.7 cells (n.s., sh-catu2) were treated with 0, 2.5, 5 or 10 μg/ml cisplatin for 6 h and analyzed for caspase 9 activity using caspase glo luminescence assay. (<b>E</b>) Cells as in (<b>D</b>) were analyzed for caspase 3/7 and (<b>F</b>) caspase 8.</p

Cell proliferation is reduced in a dose- and time dependent manner in cisplatin treated melanoma cells when α-catulin is knocked down.

<p>(<b>A</b>) Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0, 5, 10 and 20 μg/ml cisplatin for 24 h and analyzed by Western blot with antibody against Ki67. α-Tubulin was used as a loading control and quantification was performed with BioRad software. (<b>B</b>) Mel.7 cells (n.s., sh-catu2) were treated with 20, 10, 5, or 0 μg/ml cisplatin for 48 h and BrdU assay was performed. Therefore, cells were stained with BrdU solution and antibodies against BrdU and HRP conjugated secondary antibody was detected at 450 nm using a multiplate reader. (<b>C</b>) Mel.7 cells (n.s., sh-catu2) were treated with 0 or 10 μg/ml cisplatin for 18 hours, fixed, stained with propidium-iodide solution and analysed for cell cycle distribution using flow cytometry. (<b>D</b>) Cells as in (<b>C</b>) were analysed using western blot with antibodies against p21^cip/waf and p53.</p

Tandem direct repeats regulate the length of <i>Airn</i>.

<p>(A) qPCR of total (spliced+unspliced) <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a> map for location of qPCR assays). Relative <i>Airn</i> levels were set to 100% in <i>S12/+</i> cells at d14. Bars and error bars: mean and standard deviation of three differentiation sets. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test (*P = 0.1–0.5, **P = 0.001–0.01, ***P<0.001). The data show that <i>Airn</i> steady-state levels are unchanged up to 53 kb but are greatly reduced and lost at the 3′ end. (B) qPCR of spliced <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells. Details as in (A). These data show that the TDR deletion does not affect <i>Airn</i> splicing suppression but leads to a shortening at the 3′ end. (C) qPCR of unspliced <i>Airn</i> in 12.5–13.5 dpc mouse embryos confirms the significant loss of <i>Airn</i> steady-state levels at the 3′ end as seen in differentiated ES cells (A,B). Embryos from 3 litters were assayed carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+</i>/<i>TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Samples of the same genotype were averaged and the horizontal lines and error bars show mean and standard deviation. Values for individual embryos are plotted as single data points. The number of samples is given below the genotype (n). Relative <i>Airn</i> levels were set to 100% for <i>+/+</i>, all others are displayed relative to it. Samples were compared to <i>+/+</i> using an unpaired t-test. Details as (A).</p