NF-κB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells

BACKGROUND: The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1) is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2) and calsarcin-3 (CS-3) is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated. RESULTS: Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-κB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-κB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1. CONCLUSION: Our present data suggest that NF-κB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation

Influence of acrylamide on ROS, Hsp27 and NF-kB in bone marrow mesenchymal stem cells

The bone marrow mesenchymal stem cells (BM-MSCs) treated with acrylamide (ACR) were used to make out the immune response to ROS, interleukin-8 and phosphorylated Hsp27 of ACR. ACR was reported as a probable human carcinogen, neurotoxic and mutagenic. BMMSCs have the capability of immunoregulation, and participate in the process of multiple immune response. It has attracted the attention of researchers that these cells have priority to move to the damaged tissue, as a kind of potential therapeutic tool for tissue repair. ACR and BMMSCs are related to immune reactions, especially those involving in tumours and cancers. However, the interaction between ACR and BMMSCs is still poorly understood. In present study, we report the influence of ACR on BMMSCs. At first, BMMSCs were disposed with 0.5mM ACR for 72 h, and then the secretion of ROS, interleukin-8, phospho- Hsp27 and NF-kB activities, apoptosis and cell cycle, respectively, were determined. The results showed that the secretion of ROS, interleukin-8 and phosph-Hsp27 increased and NF-kB was activated, while the apoptosis and cell cycle have no obvious alteration. In conclusion, ACR probably activated the NF-kB pathway in BMMSCs via oxidative stress, which may provide new insights to study the immune response and the influence mechanism of ACR

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation

Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JHregulated insect reproduction

Differential gene expression profiles in JH-deprived and methoprene-exposed fat bodies.

<p>(A) Number of up-regulated (red) and down-regulated (blue) gene transcripts in JH-deprived fat bodies further treated with methoprene for 24 h. Fold change ≥2 and <i>P</i><0.05 were used as the cutoff criteria. (B) Significantly enriched gene ontology terms associated with up-regulated genes. (C) Significantly enriched KEGG pathways (top 10) of up-regulated genes. (D) Validation of RNA-seq data by qRT-PCR for genes associated with DNA replication. Fold change was calculated as: mRNA levels in methoprene-treated females/mRNA levels in precocene-treated females.</p

Responsiveness of <i>Mcm4</i> and <i>Mcm7</i> to JH and <i>Met</i> RNAi.

<p>(A) Relative mRNA levels of <i>Mcm4</i> and <i>Mcm7</i> in the fat body of adult females treated with precocene (P10d) and those further treated with methoprene for 6, 12, 24 and 48 h (PM6h, PM12h, PM24h and PM48h, respectively). mRNA levels in precocene-treated fat bodies were used as the calibrator. *, <i>P</i><0.05, **, <i>P</i><0.01 and ***, <i>P</i><0.001 compared to P10d; n = 4–6. (B) <i>Mcm4</i> and <i>Mcm7</i> mRNA abundance in fat bodies of female locusts from 0 to 10 days post adult eclosion (PAE). *, <i>P</i><0.05 and **, <i>P</i><0.01 compared to PAE0; n = 4–6. (C) Relative levels of <i>Mcm4</i> and <i>Mcm7</i> transcripts in fat bodies of dsGFP control (iGFP), <i>Met</i>-RNAi (iMet), and iMet further treated with methoprene for 48 h (iMet+JHA). *, <i>P</i><0.05; n = 10–12. (D) Alignment of DNA element sequences containing E-box and E-box-like motifs in the upstream promoter regions of locust <i>Mcm7</i> and <i>Mcm4</i> with experimentally tested Met-binding consensus sequences except that of <i>DmKr-h1</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Kayukawa1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Li1" target="_blank">[9]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Zou1" target="_blank">[34]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Cui1" target="_blank">[36]</a>. (E) EMSA using the Mcm4 probe listed in (D) with fat body nuclear extracts of iGFP and iMet (shown is a representative short exposure; longer exposures in some experiments showed additional two bands of less interest; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#s2" target="_blank">Results</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702.s007" target="_blank">Fig. S7</a>). (F) EMSA using Mcm7 probe listed in (D) and with fat body nuclear extracts of iGFP and iMet. For both (E) and (F), arrows indicate the most likely specific bands. FP, free probe.</p

<i>Mcm4</i> and <i>Mcm7</i> transcription and the JH-receptor complex.

<p>(A) Upper panels: western blot (WB) showing the expression of Flag-Met^1-3108 and V5-SRC in S2 cells; lower panel: immunoprecipitation (IP) showing the interaction of Flag-Met^1-3108 and V5-SRC in the presence of 10 µM JH III or methoprene. α-Flag, Flag antibody; α-V5, V5 antibody. (B) Luciferase assays after co-transfection of pGL4.10/<i>Mcm4</i>^{−933 to −37} or pGL4.10/<i>Mcm7</i>^{−933 to −11} into S2 cells compared with the pAc5.1 empty vector, pAc5.1/Flag-Met^1-3108 (Met) or/and pAc5.1/V5-SRC (SRC), with or without 10 µM JH III or methoprene (JHA) treatment. (C) EMSA using the Mcm4 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe. (D) EMSA using the Mcm7 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe.</p