Suppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation

Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression

kdm4aa is required for reproduction and development of zebrafish

Lysine-specific demethylase 4A (KDM4A) catalyzes demethylation of histone lysine residues, which regulates chromatin state and transcription. In drosophila and mice, KDM4A plays an important role in multiple biological processes including development, aging, metabolism, and immunity, however the functions of KDM4A in fish are still unclear. There are two copies of the kdm4a gene in zebrafish, namely kdm4aa and kdm4ab, kdm4aa was edited using CRISPR/Cas9 technology in the present study, then homozygous kdm4aa mutants (kdm4aa−/−) were obtained, and loss of kdm4aa was confirmed by sequencing and increased H3K9me3. Whole-mount in situ hybridization showed that kdm4aa is widely expressed during the embryonic development of zebrafish. Compared with WT zebrafish, kdm4aa−/− zebrafish showed no significant difference in gamete formation and fertilization, but the survival rate of kdm4aa−/− embryos dramatically reduced to 21% at 26 hpf. Further observation showed that about 80% of survived kdm4aa−/− zebrafish experienced disruption in stripe formation, and 10% of survived kdm4aa−/− zebrafish underwent vertebral malformation. Alizarin red S staining demonstrated the abnormal spinal development in kdm4aa−/− zebrafish. These results indicated that kdm4aa is required for normal embryonic development of zebrafish, loss of kdm4aa function leads to decreased survival during the early stages of zebrafish development and morphological variation in adult zebrafish

Ferromagnetic Property and Synthesis of Onion-Like Fullerenes by Chemical Vapor Deposition Using Fe and Co Catalysts Supported on NaCl

Metal-encapsulating onion-like fullerenes (M@OLFs) were synthesized by CVD at relatively low temperature (420C∘) using Fe and Co nanoparticles impregnated into NaCl as catalyst. The morphology and structure of the products were characterized by field emission scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. The results show that Fe@OLFs and Co@OLFs with stacked graphitic fragments were prepared using Fe/NaCl or Co/NaCl as catalysts; after Co@OLFs were immersed in concentrated HCl for 48 hours, Co nanoparticles encapsulated by carbon shells were not removed, meaning that carbon shells can protect the encapsulated Co cores against environmental degradation. The coercivity value (750.23 Oe) of Co@OLFs showed an obvious magnetic property

Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells.

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish

Promotion of Melanoma Cell Proliferation by Cyclic Straining through Regulatory Morphogenesis

The genotype and phenotype of acral melanoma are obviously different from UV-radiation-induced melanoma. Based on the clinical data, mechanical stimulation is believed to be a potential cause of acral melanoma. In this case, it is desirable to clarify the role of mechanical stimulation in the progression of acral melanoma. However, the pathological process of cyclic straining that stimulates acral melanoma is still unclear. In this study, the influence of cyclic straining on melanoma cell proliferation was analyzed by using a specifically designed cell culture system. In the results, cyclic straining could promote melanoma cell proliferation but was inefficient after the disruption of cytoskeleton organization. Therefore, the mechanotransduction mechanism of promoted proliferation was explored. Both myosin and actin polymerization were demonstrated to be related to cyclic straining and further influenced the morphogenesis of melanoma cells. Additionally, the activation of mechanosensing transcription factor YAP was related to regulatory morphogenesis. Furthermore, expression levels of melanoma-involved genes were regulated by cyclic straining and, finally, accelerated DNA synthesis. The results of this study will provide supplementary information for the understanding of acral melanoma

Impact of TAM67 on AP1 factors in vivo.

<p>TAM67-rTA mice were treated with (+) or without (−) 2 mg/ml doxycycline in drinking water for 3 days. <b>A</b> Murine epidermis was collected free of the dermis by high temperature separation as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Rorke1" target="_blank">[35]</a>. Total extract was prepared for immunoblot to detect the indicated proteins. TAM67-FLAG was detected with anti-FLAG. <b>B</b> Interaction of TAM67 with AP1 site consensus element. Nuclear extracts were prepared from epidermis and incubated with AP1c-P³² and other probes as indicated. FP indicates free probe, NE indicates nuclear extract. Similar results were observed in each of three experiments. <b>C</b> Impact of TAM67 on interaction of endogenous AP1 factors with AP1 site element. Nuclear extracts were prepared from TAM67-negative and TAM67-expressing epidermis and incubated with the AP1c-P³² and antibodies as indicated. The complexes were then separated on a non-denaturing 6% polyacrylamide gel. FP indicates free probe and NE is nuclear extract. Note the reduction in jun factor binding in the presence of TAM67-FLAG (left panel). We did not observe a significant reduction in fos factor interaction in the presence of TAM67 (right panel).</p

TAM67-FLAG inhibits hINV gene expression.

<p><b>A</b> TAM67 reduces hINV protein and mRNA level. Keratinocytes were infected with indicated MOI of tAd5-EV or tAd5-TAM67-FLAG and after 48 h extracts were prepared to detect hINV protein by immunoblot and mRNA by quantitative PCR. The values are mean ± SD and the asterisks indicate a significant reduction using student’s t-test, n  = 3 (p<0.001). <b>B</b> TAM67 suppresses AP1 factor-dependent promoter activity. Keratinocytes were transfected with the indicated hINV reporter constructs in the presence of empty pcDNA3 vector or pcDNA3-TAM67-FLAG and treated 24 h with or without 50 ng/ml TPA prior to preparation of extracts and assay of luciferase activity. The values are mean ± SEM and the asterisks indicate a significant reduction using student’s t-test, n = 3 (p<0.001).</p

TAM67 binds to the AP1-5 site of hINV gene promoter.

<p>Keratinocytes were infected with 10 MOI tAd5-EV or tAd5-TAM67-FLAG and after 24 h nuclear extracts were prepared for gel shift. <b>A</b> TAM67 interaction with hINV promoter AP1-5 site. Nuclear extracts were incubated with AP1-5-P³² with or without a 50-fold molar excess of AP1-5 or AP1-5 m oligonucleotide, or anti-FLAG antibody, and electrophoresed on a 6% acrylamide non-denaturing gel. FP indicates free probe and NE is nuclear extract. The arrow indicates the major shifted bands and asterisks indicate supershifted bands. AP1-5 is an oligonucleotide encoding the AP1-5 site of hINV promoter. AP1-5 m is an AP1-5 mutant that does not bind AP1 transcription factors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Welter1" target="_blank">[47]</a>. <b>B</b> TAM67 inhibits AP1 factor interaction with AP1-5. Nuclear extracts were incubated with AP1-5-P³² in the absence or presence of c-jun, junB, junD, Fra-1, Fra-2, c-fos, or fosB specific antibodies, and electrophoresed on a 6% acrylamide non-denaturing gel. Arrows indicate major shifted band and asterisks indicate supershifted bands. FP is free probe. <b>C</b> ChIP analysis reveals TAM67 presence at the hINV upstream regulatory region AP1-5 site <i>in vivo</i>. Nuclear extracts were prepared for ChIP analysis and incubated with anti-IgG or anti-FLAG and the precipitated DNA was analyzed for AP1-5 site encoding sequences. The values are mean ± SD (n = 3, p<0.001) and the asterisk indicates a significant increase compared to all other groups. Nucleotides −2218/−2055 encodes the AP1-5 site and nucleotides −1040/−919 is a region of the hINV upstream regulatory region that lacks an AP1 site.</p

TAM67 suppresses c-jun promoter activity.

<p><b>A</b> TAM67 reduces c-jun mRNA. Keratinocytes were infected with empty (EV) or TAM67-FLAG encoding adenovirus (10 MOI) and after 24 h mRNA was prepared and c-jun mRNA level was measured by quantitative PCR. <b>B</b> TAM67 suppresses c-jun promoter activity. Keratinocytes were transfected with 1 µg of the indicated c-jun promoter luciferase reporter construct and 1 µg of pcDNA3 (EV) or pcDNA3-TAM67-FLAG (TAM67-FLAG). After 24 h the cells were harvested and assayed for luciferase activity. The values in both plots are mean <u>+</u> SD and the asterisks indicate a significant reduction (p<0.005, n = 3). <b>C</b> Map of c-jun promoter region. The promoter constructs encode nucleotides −1780 to +731 with the transcription start site at +1. c-jun(−1780/+731) is the wild-type intact promoter and c-jun(−1780/+731)-AP1m is a construct in which the critical AP1 sites are eliminated by mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Wei1" target="_blank">[48]</a>. LUC indicates the luciferase gene. The numbers are given in nucleotides.</p