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Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α 8 β 1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway., (© 2021. The Author(s).)

We investigated whether docosahexaenoic acid (DHA), a dietary n-3 fatty acid, modulates calcium (Ca 2+ ) signaling and cell cycle progression in human Jurkat T-cells. Our study demonstrates that DHA inhibited Jurkat T-cell cycle progression by blocking their passage from S phase to G2/M phase. In addition, DHA decreased the plasma membrane expression of TRPC3 and TRPC6 calcium channels during T-cell proliferation. Interestingly, this fatty acid increased plasma membrane expression of TRPC6 after 24 h of mitogenic stimulation by phorbol-13-myristate-12-acetate (PMA) and ionomycin. These variations in the membrane expression of TRPC3 and TRPC6 channels were not directly correlated with the mRNA expression, indicating that it was a post-translational phenomenon. DHA increased free intracellular calcium concentrations, [Ca 2+ ]i, via opening TRPC3 and TRPC6 channels. We conclude that the anti-proliferative effect of DHA might involve the modulation of TRPC3 and TRPC6 channels in human T-cells., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest whatsoever., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

An active metabolite of vitamin A, all-trans retinoic acid (ATRA), is known to exert immunomodulatory functions. This study investigates the possible immune potentiating effect of ATRA on NF-κB activity in human monocytic THP-1 cells after exposure to unmethylated CpG DNA ODN2006. We observed that challenge with ODN2006 significantly enhanced the NF-κB activity of PMA-differentiated THP-1 cells. ATRA synergistically enhanced NF-κB activity of cells, in a concentration- and time-dependent manner. The enhanced NF-κB activity of PMA-differentiated THP-1 cells after ODN2006 challenge was dependent on the RAR/RXR pathway. To determine the mechanism involved in increasing in the NF-κB activity of stimulated THP-1 cells, we examined the effects of PMA and ATRA on the expression of TLR9 (a receptor of ODN2006) in THP-1 cells. PMA treatment significantly enhanced both the intracellular and cell surface expression of TLR9, while ATRA alone showed no effect. However, ATRA synergistically enhanced the cell surface TLR9 expression of PMA-differentiated cells. To determine whether the ATRA-enhanced NF-κB activity is due to the enhanced cell surface TLR9 expression, we examined NF-κB activity after treatment with anti-TLR9 blocking antibody. Results revealed that the anti-TLR9 antibody treatment almost completely reverses the ATRA-enhanced NF-κB activity, suggesting that ATRA enhances NF-κB activity through upregulation of the cell surface TLR9 expression in PMA-differentiated and unmethylated CpG challenged THP-1 cells.

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of Enterobacteriaceae , raising serious global health concerns. In this paper, we used recombinant mcr-1 -expressing Escherichia coli to study the impact of MCR-1 products on E. coli -induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant mcr-1 -expressing E. coli significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1β compared with mcr-1 -negative strains. Caspase-1 activity and IL-1β secretion were significantly less activated by mcr-1 -positive E. coli strains than the mcr-1 -negative parental strain. Similar results were obtained with clinical isolates of mcr-1 -positive E. coli , suggesting that, in addition to colistin resistance, the expression of mcr-1 allows the escape of early host innate defenses and may promote bacterial survival., (Copyright © 2020 American Society for Microbiology.)

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Eomesodermin (Eomes) is a member of T-box transcription factor family and plays an important role in the regulation of a wide variety of developmental processes and immune response in animals. Here we report cloning and characterization of the full-length cDNA of Atlantic cod Eomes (GmEomes), which possesses a TBOX_3 domain similar to its counterpart in mammals. The regulated expression was observed in head kidney and spleen in response to live Vibrio anguillarum infection in vivo, and spleen leukocytes in vitro after PMA and poly I:C stimulation. Furthermore, we determined a 694 bp sequence, upstream of the transcriptional start site (TSS), to contain a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. The results showed that the basal promoters and positive transcriptional regulator activities of GmEomes were dependent by sequences located from -694 to -376 bp upstream of TSS. Furthermore, we found that some Eomes binding sites were present in the 5'-flanking regions of the cod IFNγ gene predicted by bioinformatics. However, Co-transfection of eomesodermin overexpression plasmids with INFγ reporter vector into CHSE-214 cells determined that Atlantic cod eomesodermin played a minor role in activation of the INFγ promoter., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Interleukin 16 is an immunomodulatory chemokine that signals through CD4 + T cells, monocytes, macrophages and dendritic cells. Its expression in immune-related cells enhances the antimicrobial effect and inhibits HIV replication in macrophages. However, the role of IL-16 in macrophage polarization is uncertain. Mir-145 was reported to regulate IL-10 expression by targeting histone deacetylase 11 and promotes alternatively activated macrophage (M2) polarization. Mir-145 was also predicted to target IL-16 mRNA. We aimed to explore the roles of IL-16 and mir-145 in macrophage polarization and antimicrobial functions., Methods: THP1 monocytes were employed in this study, and their cell activity when incubated with different concentrations of IL-16 was evaluated using the CCK-8 cell counting kit. To obtain polarized macrophages, THP-1 cells were induced by IL-4 and IL-13 following PMA incubation (M2 polarized macrophages) or induced by IFN-gamma and LPS (M1 classical macrophage activation). The influence of IL-16 on macrophage phagocytosis was quantified by the amount of chicken red blood cell phagocytized. IL-16, IL-10 and miR-145 expression in THP1 monocytes and induced macrophages was quantified by quantitative PCR. The miR-145 and IL-16 targeting relationship was verified by the dual luciferase reporter assay. The influence of IL-16 and mir-145 on macrophage polarization was evaluated by M1 and M2 macrophage characterized marker gene expression., Results: The M0 macrophage subtype was induced by PMA. The M1 and M2 subtypes of macrophage were successfully induced by M1- and M2-specific induction. M1 macrophages express higher levels of IL-16 than M2 macrophages but express lower levels of IL-10 and mir-145 than M2 cells. IL-16 with a concentration up to 150 ng/mL has no influence on THP-1 cell proliferation but improves macrophage phagocytosis ability with the down-expression of IL-10 and up-expression of pro-inflammatory cytokines such as IL-1a and IL-6. Knockdown with its target siRNA is beneficial for macrophage maintenance but reduces phagocytosis ability. Mir-145 specifically targets the IL-16 3'UTR verified by the dual luciferase reporter assay. Mir-145 downregulates IL-16 expression and upregulates IL-10 expression, thereby promoting M2 macrophage polarization., Conclusion: IL-16 modulates macrophage polarization through regulating IL-10, IL-1a and IL-6 expression. Mir-145 is involved in M2 macrophage polarization by targeting IL-16 and enhancing IL-10 expression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The main aim of this investigation was to study the regulatory roles of let-7b and miR-155-3p on the expression of inflammation-associated genes in monocytes, macrophages, and lipopolysaccharide (LPS)-activated macrophages (AcM). A second goal was to analyze the potential modulatory roles of different fatty acids, including oleic, palmitic, eicosapentaenoic (EPA), and docosahexaenoic (DHA), on the expression of these miRNAs in the three cell types. This hypothesis was tested in human acute monocytic leukemia cells (THP-1), which were differentiated into macrophages with 2-O-tetradecanoylphorbol-13-acetate (TPA) and further activated with LPS for 24 h. Monocytes, macrophages, and AcM were transfected with a negative control, or mimics for miR-155-3p and miR-let-7b-5p. The expression of both miRNAs and some proinflammatory genes was analyzed by qRT-PCR. Interestingly, let-7b mimic reduced the expression of IL6 and TNF in monocytes, and SERPINE1 expression in LPS-activated macrophages. However, IL6, TNF, and SERPINE1 were upregulated in macrophages by let-7b mimic. IL6 expression was higher in the three types of cells after transfecting with miR-155-3p mimic. Similarly, expression of SERPINE1 was increased by miR-155-3p mimic in monocytes and macrophages. However, TLR4 was downregulated by miR-155-3p in monocytes and macrophages. Regarding the effects of the different fatty acids, oleic acid increased the expression of let-7b in macrophages and AcM and also increased the expression of miR-155 in monocytes when compared with DHA but not when compared with non-treated cells. Overall, these results suggest anti- and proinflammatory roles of let-7b and miR-155-3p in THP-1 cells, respectively, although these outcomes are strongly dependent on the cell type. Noteworthy, oleic acid might exert beneficial anti-inflammatory effects in immune cells (i.e., non-activated and LPS-activated macrophages) by upregulating the expression of let-7b.

The rupture of atherosclerotic plaques may result in the formation of thrombi, which may induce subsequent cardiac events such as acute myocardial infarction. Overproduction of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducers (EMMPRINs) by monocytes and macrophages may lead to rupture of atherosclerotic plaques as a result of the degradation of the extracellular matrix. The purinergic 2X7 receptor (P2X7R) is expressed in macrophages that are assembled in atherosclerotic lesions of human carotid arteries. P2X7R may serve a crucial role in the development of atherosclerosis; therefore, the present study aimed to determine whether P2X7R regulated the expression of EMMPRIN and MMP‑9 in phorbol 12‑myristate 13‑acetate (PMA)‑induced macrophages. In addition, the potential molecular mechanisms involved in this process were investigated. THP‑1 human monocytic cells were pretreated with A‑438079 (a specific inhibitor of P2X7R) for 1 h and subsequently incubated with or without PMA for 48 h. Exposure to A‑438079 significantly decreased the expression of MMP‑9 and EMMPRIN in the PMA‑induced macrophages and attenuated the activation (phosphorylation) of mitogen‑activated protein kinase (MAPK) signaling, including c‑Jun N‑terminal kinase, p38 and extracellular signal‑regulated kinase. The present study also demonstrated that 5'‑AMP‑activated protein kinase (AMPK) was activated by PMA exposure during differentiation from monocytes to macrophages. This activation was reversed by A‑438079 treatment through the inhibition of P2X7R expression. These results suggested that the inhibition of P2X7R may be able to suppress the AMPK/MAPK signaling pathway and consequently downregulate both EMMPRIN and MMP‑9 expression in PMA‑induced macrophages.

Neutrophils undergo cell death processes once their physiological function has been fulfilled. Apoptosis, necrosis, pyroptosis, or NETosis, a unique form of cell death, could occur, depending on the type of stimulant or inhibitory intervention. We investigated whether phorbol myristate acetate (PMA) and Klebsiella pneumoniae (KP), serving as stimulants, or whether an inhibitor (cytochalasin B, CytB) could alter the morphology and gene expression pattern associated with mouse neutrophil cell death. Fluorescence microscopy, flow cytometry, and real-time PCR approaches were used to identify morphological changes, percentages of cell death, and gene expression patterns, respectively. The results showed an increase in the percentage of cell death in PMA and KP groups, whereas CytB exerted inhibitory effects. Fluorescently stained cell nuclei revealed significantly different percentages of cell death among treatments. Moreover, there was a stepwise increase in cell death from 90 to 150 min after stimulation. Quantification of the release of neutrophil extracellular traps (NETs) demonstrated clearly elevated amounts in cells stimulated with KP, while the amount of NETs was slightly increased or unchanged with PMA or CytB treatments. Analysis of the genes involved in cell death revealed that mRNA expression of CASP1, IL1β, IL18, MCL1, NLRP3, and PYCARD was down-regulated in the PMA group, with the exception of AIM2 and CASP3. The expression of AIM2, IL1β, MCL1, and NLRP3 was up-regulated in KP-stimulated neutrophils, while CASP1, CASP3, IL18, and PYCARD genes were down-regulated. In summary, a spectrum of specific cell stimulants and exposure durations accounted for different outcomes in mouse neutrophil cell death.

The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LβT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LβT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Neutrophils are essential innate immune cells whose responses are crucial in the clearance of invading pathogens. Neutrophils can respond to infection by releasing neutrophil extracellular traps (NETs). NETs are formed of chromatin and specific granular proteins and are released after execution of a poorly characterized cell death pathway. Here, we show that NET formation induced by PMA or Candida albicans is independent of RNA polymerase II and III-mediated transcription as well as of protein synthesis. Thus, neutrophils contain all the factors required for NET formation when they emerge from the bone marrow as differentiated cells.

Cordycepin (3'-deoxyadenosine) is one of the active components isolated from Cordyceps militaris, and has been shown to have anti-inflammatory, anti-oxidant, anti-aging, and anti-cancer effects. Mast cell-derived thymic stromal lymphopoietin (TSLP) plays an important role in the pathogenesis of allergic inflammatory reactions. Here, we investigated the regulatory effect and mechanisms of cordycepin on the expression of TSLP in the human mast cell line, HMC-1 cells, and in the human keratinocyte cell line, HaCaT cells. Cordycepin significantly decreased the production and mRNA expression of TSLP through the inhibition of caspase-1 and nuclear factor-κB activation. Cordycepin also significantly reduced the phosphorylation of receptor-interacting protein 2 and inhibitory kappa B (IκB) kinase β. Cordycepin significantly decreased the production and mRNA expression of interleukin (IL)-8, IL-1β, IL-6, and tumor necrosis factor-α in activated HMC-1 cells. Moreover, cordycepin significantly decreased the levels of TSLP in activated HaCaT cells. Our studies suggest that cordycepin can be applied to the treatment of allergic inflammatory diseases exacerbated by TSLP.

Progranulin (PGRN) is a multi-functional protein known to be involved in inflammation. Recent studies have found that PGRN has dual roles in inflammation and exerts anti-inflammatory and pro-inflammatory function in different diseases. However, the role of PGRN in psoriasis has not been fully elucidated. Here, we detected preferential expression of PGRN in human psoriatic lesions and serum. Moreover, serum PGRN/tumour necrosis factor-α ratio was negatively correlated with disease severity. To investigate the role of PGRN in the pathogenesis of psoriasis, we used wild-type (WT) and PGRN(-/-) mice in a model of 12-O-tetradecanoylphorbol 13-acetate (TPA) -induced psoriasis-like inflammation. We demonstrated that PGRN expression was dramatically enhanced in the psoriasis-like lesions of TPA-treated WT mice, in accordance with human psoriatic lesions. Surprisingly, PGRN(-/-) mice were more sensitive to the development of TPA-induced psoriasis-like inflammation. The mechanism underlying this increased sensitivity of PGRN(-/-) mice to TPA-induced psoriasis-like inflammation was impaired differentiation of regulatory T cells in lymph nodes and decreased recruitment of these cells in the affected skin, which results in more severe inflammation. Hence, in WT mice, PGRN promotes differentiation and recruitment of regulatory T cells at the site of inflammation, which protects the skin from an exaggerated psoriasis-like inflammatory response., (© 2015 John Wiley & Sons Ltd.)

Nuclear factor κB (NFκB) signaling plays ubiquitous roles in inflammation, immune response and neurogenesis. G protein-coupled receptor kinase 5 (GRK5) can protect neurons from degeneration. GRK5 also mediates tumor necrosis factor-α (TNFα)-induced NFκB signaling through the phosphorylation of IκBα. Here, we show that NFκB signaling is involved in neural stem cell (NSC) differentiation. The IκBα/p65 pathway was activated by phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC). Once the NFκB was activated, the initial stage of neural differentiation was induced, with an increased level of GRK5 in NSCs. This finding was reversed in response to the NFκB inhibitor N-acetyl cysteine (NAC). To evaluate the effect of GRK5-NFκB signaling crosstalk on NSC neurogenesis and apoptosis, GRK5 was knocked down by siRNAs in cell culture. SiRNAs against GRK5 not only impaired neural differentiation and axogenesis, but also induced apoptosis of NSC. GRK5 knockdown affected the transcription of NFκB, phosphorylation of the liver kinase B1 (LKB1) and the activity of caspase 3, thereby modulated neurogenesis and apoptosis. Taken together, our findings reveal a novel function of GRK5 in neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

G-protein coupled receptor 43 (GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis. Here, we present the first full characterization of the GPR43 promoter and 5'-UTR. 5'-RACE of the GPR43 transcript identified the transcription start site (TSS) and a 124 bp 5'-UTR followed by a 1335 bp intron upstream of the ATG start codon. The sequence spanning -4560 to +68 bp relative to the GPR43 TSS was found to contain strong promoter activity, increasing luciferase reporter expression by >100-fold in U937 monocytes. Stepwise deletions further narrowed the putative GPR43 promoter (-451 to +68). Site-directed mutagenesis identified XBP1 as a core cis element, the mutation of which abrogated transcriptional activity. Mutations of predicted CREB, CHOP, NFAT and STAT5 binding sites, partially reduced promoter activity. ChIP assays confirmed the binding of XBP1 to the endogenous GPR43 promoter. Consistently, GPR43 expression is reduced in monocytes upon siRNA-knockdown of XBP1, while A549 cells overexpressing XBP1 displayed elevated GPR43 levels. Based on its ability to activate XBP1, we predicted and confirmed that TNFα induces GPR43 expression in human monocytes. Altogether, our findings form the basis for strategic modulation of GPR43 expression, with a view to regulate GPR43-associated diseases.

We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells). We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c-KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.

The immediate early gene c-Fos is reported to be regulated by Elk-1 and cAMP response element-binding protein (CREB), but whether nuclear factor (NF)-κB is also required for controlling c-Fos expression is unclear. In this study, we determined how NF-κB's coordination with Elk/serum response factor (SRF) regulates c-fos transcription. We report that PMA strongly induced c-Fos expression, but tumor necrosis factor (TNF)-α did not. In mouse embryonic fibroblasts, the PMA induction of c-Fos was suppressed by a deficiency in IKKα, IKKβ, IKKγ, or p65. By contrast, in human embryonic kidney 293 cells, PMA induced c-Fos independently of p65. In accordance with these results, we identified an NF-κB binding site in the mouse but not human c-fos promoter. Under PMA stimulation, IKKα/β mediated p65 phosphorylation and the binding of the p65 homodimer to the NF-κB site in the mouse c-fos promoter. Furthermore, our studies demonstrated independent but coordinated functions of the IKKα/β-p65 and extracellular signal-regulated kinase (ERK)-Elk-1 pathways in the PMA induction of c-Fos. Collectively, these results reveal the distinct requirement of NF-κB for mouse and human c-fos regulation. Binding of the p65 homodimer to the κB site was indispensable for mouse c-fos expression, whereas the κB binding site was not present in the human c-fos promoter. Because of an inability to evoke sufficient ERK activation and Elk-1 phosphorylation, TNF-α induces c-Fos more weakly than PMA does in both mouse and human cells.

Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2), activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph) coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1), by phosphorylating H3S10 at its 5' regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

This study was carried out to explore the molecular mechanism for down-regulation of TRPC6 expression in the reactive oxygen species (ROS)/PKC signaling in kidney cells. In cultured human mesangial cells, H2O2 and TNF-α inhibited TRPC6 mRNA expression in a time-dependent manner. Inhibition of NF-κB reversed both H2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκBα using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. a Ca(2+) imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca(2+) entry, respectively. Further experiments showed that PMA, but not its inactive analog 4α-phorbol 12, 13-didecanoate (4α-PDD), caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by Gö6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented an H2O2 inhibitory effect on TRPC6 protein expression, suggesting p65 as a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased the association of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented a H2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.

The current study was designed to determine if the NADPH-oxidase NOX2 plays a role in development of obesity after high fat feeding. Wild-type (WT) mice and mice lacking the essential cytosolic NOX2 system component p47(phox) (P47KO mice) were fed AIN-93G diets or high-fat diets (HFD) containing 45% fat and 0.5% cholesterol for 13 wk from weaning. Fat mass was increased to a similar degree by HFD in males of both genotypes (P < 0.05). However, female P47KO-HFD mice had no increase in adiposity or adipocyte size relative to female WT-HFD mice. Resistance to HFD-driven obesity in P47KO females was associated with increased expression of hepatic TFAM and UCP-2 mRNA, markers of mitochondrial number and uncoupling, and increased expression of hepatic mitochondrial respiratory complexes and whole body energy expenditure in response to HFD. Microarray analysis revealed significantly lower expression of mRNA encoding genes linked to energy metabolism, adipocyte differentiation (PPARγ), and fatty acid uptake (CD36, lipoprotein lipase), in fat pads from female P47KO-HFD mice compared with WT-HFD females. Moreover, differentiation of preadipocytes ex vivo was suppressed more by 17β-estradiol in cells from P47KO compared with cells from WT females in conjunction with overexpression of mRNA for Pref-1 (P < 0.05). HFD mice of both sexes were resistant to the development of hyperglycemia and hepatic steatosis (P < 0.05) and had reduced serum triglycerides, leptin, and adiponectin relative to WT-HFD mice (P < 0.05). These data suggest that NOX2 is an important regulator of metabolic homeostasis and diet-induced obesity.

Interleukin (IL)-13 is a cytokine produced by activated CD4(+) T cells that plays a critical role in promoting allergic responses and tumor cell growth. The 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a natural ligand for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), a known regulator of anti-inflammatory activities. We determined the effects of 15d-PGJ(2) on IL-13 expression in the Jurkat E6.1 T-cell line and in peripheral blood mononuclear cells. Semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay revealed that treatment of activated T cells with 15d-PGJ(2) significantly decreased IL-13 mRNA transcription and secretion, respectively. This inhibition by 15d-PGJ(2) was independent of PPAR-γ since treatment with GW9662, an irreversible antagonist of the nuclear receptor, produced no effect. Our data also revealed the involvement of nuclear factor-κB in mediating 15d-PGJ(2)-dependent down regulation of IL-13 expression. Collectively, these results demonstrate the potential of 15d-PGJ(2) in attenuating expression and production of IL-13 in activated T cells., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Background/aims: Multidrug resistance (MDR) is a critical issue during chemotherapy of cancers. Phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, is a typical activator of protein kinase C (PKC). In the present study, we investigated the effect of PMA on MDR and P-glycoprotein (P-gp) gene expression in K562/ADM cells., Methods: 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay was used to assess adriamycin (Adr)-induced cytotoxicity towards K562/ADM cells in the absence or presence of PMA. The intracellular accumulation of Adr was measured by determining the mean fluorescence intensity. The effect of PMA on P-gp activity was investigated by rhodamine-123 accumulation and efflux experiment. Protein expression and mRNA expression of P-gp in K562/ADM cells were determined by Western blot analysis and real-time qPCR, respectively., Results: Adr-induced cytotoxicity towards K562/ADM cells was significantly decreased by PMA at 5 μmol/l. Furthermore, intracellular Adr-associated mean fluorescence intensity was attenuated by 53.8% 1 h after exposure to PMA at 5 μmol/l compared with the control group (p < 0.05). A dose-dependent decrease of intracellular rhodamine-123 and increase of efflux activity of P-gp were also observed in K562/ADM cells incubation with PMA. In addition, P-gp mRNA and protein expression were significantly induced by PMA., Conclusion: Activation of PKC pathway by PMA can significantly induce expression and activity of P-gp, and thus decrease intracellular Adr level and strengthen MDR in K562/ADM cells., (© 2013 S. Karger AG, Basel.)

The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

Gonadotropin-releasing hormone (GnRH) plays a major role in the hypothalamic-pituitary-gonadal (HPG) axis, and synthesis and secretion of GnRH are regulated by gonadal steroid hormones. Disruptions in androgen levels are involved in a number of reproductive defects, including hypogonadotropic hypogonadism and polycystic ovarian syndrome. Androgens down-regulate GnRH mRNA synthesis in vivo and in vitro via an androgen receptor (AR)-dependent mechanism. Methyltrienolone (R1881), a synthetic AR agonist, represses GnRH expression through multiple sites in the proximal promoter. In this study, we show AR also represses GnRH transcription via the major enhancer (GnRH-E1). A multimer of the -1800/-1766 region was repressed by R1881 treatment. Mutation of two bases, -1792 and -1791, resulted in decreased basal activity and a loss of AR-mediated repression. AR bound to the -1796/-1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region. We conclude that AR repression of GnRH-E1 acts via multiple AR-responsive regions, including the site at -1792/-1791., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCε inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCε, and this interaction was totally inhibited by the PKCε inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCε and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCε and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.

Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.

Macrophage-derived chemokine (CCL22) is a member of the CC-family of chemokines and is synthesized by monocyte-derived macrophages and dendritic cells (DCs). In this study, we investigate the relationship between monocytes/macrophages and histamine in atherosclerosis and discover that histamine levels regulate various immunologically important molecules and influences atherosclerotic progression. Immunohistochemical analysis of human atherosclerotic lesions revealed that macrophages and DCs express CCL22. The human acute monocytic leukemia cell line (THP-1) adhered to culture plates and morphologically changed to macrophage-like cells when treated with tetradecanoylphorbol-13-acetate (TPA). Macrophage-like cells derived from THP-1 cells and cultivated peripheral blood mononuclear cells (PBMCs) show similar expression of CCL22. Gene expression of CCL22 was also detected in THP-1 cells treated with histamine and the expression of the protein produced by the CCL22 gene is similar in PBMCs and THP-1 cells. In addition, the histamine H2 receptor mediated these reactions. Our results suggest that CCL22 expression in monocytes is regulated by histamine, and that CCL22 is involved centrally in the development of human atherosclerotic lesions. In conclusion, CCL22 is a marker that is a characteristic of the monocytes/ macrophages migrating into atherosclerotic lesions and histamine plays a role in regulating its expression., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Background: Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites., Results: Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death., Conclusions: Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

BAFF is associated with various immunological diseases. Previously, we have reported that mouse B cell activating factor (mBAFF) expression was dependent on nuclear localization of co-activator, p300 and the activation of transcription factors including NF-κB and CREB. Here, we investigated whether transcription factor, c-Fos, regulates human (h) BAFF expression through promoter activation by PMA-induced reactive oxygen species (ROS) production. We cloned hBAFF promoter into luciferase-expressing pGL3-basic vector. The activity of 1.0 kb hBAFF promoter was higher than that in 0.75, 0.5 or 0.25 kb hBAFF promoter. The existence of three AP-1 binding motifs was computer-analyzed in hBAFF promoter. The stimulation with PMA and ionomycin (IOM) increased 1.0 kb hBAFF promoter activity, time-dependently. PMA/IOM-stimulation rapidly enhanced c-Fos expression in THP-1 human pro-monocytic cells. Binding of c-Fos to hBAFF promoter was detected by chromatin immunoprecipitation (ChIP) assay. hBAFF expression and its promoter activity were decreased by the transfection with small interference (si) RNA of c-Fos. ROS production in THP-1 cells was increased by PMA/IOM-stimulation. In addition, hBAFF activity stimulated by PMA/IOM was reduced by N-acetyl-cysteine (NAC), a well-known ROS scavenger. Serum starvation (0.5% FBS) producing ROS and the exogenous H(2)O(2) treatment also enhanced hBAFF promoter activity. c-Fos expression and AP-1 binding to oligonucleotide were reduced by the treatment with NAC. H(2)O(2) was not able to induce hBAFF expression in the presence of staurosporine, PKC inhibitor. Data suggest that hBAFF expression could be regulated by promoter activation through c-Fos association, which might be dependent on PMA-induced ROS production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ultraviolet A (UVA) rays penetrate the dermis, influencing the function of different cells, including mast cells, able to produce angiogenic factors. We investigated vascular endothelial growth factor (VEGF) release and gene-expression from mast cells (MCs), after UVA irradiation in vitro. The release of VEGF-A by Human MCs-1 (HMC-1) was induced by calcium ionophore A23187 and phorbol 12 myristate 13 acetate (PMA). Half of the cells received increasing doses of UVA (5, 25 and 50 J/cm(2)), the unirradiated HMC-1 served as controls. VEGF release and VEGF's messenger ribonucleic acid (mRNA) were detected respectively by enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results showed a UVA dose-dependent inhibitory effect on VEGF-A release from HMC-1. In particular, the release ability was reduced by 71.2% with 5 J/cm(2); 85% with 25 J/cm(2) and 86.3% with 50 J/cm(2). The VEGF-A RNA expression was reduced after UVA irradiation with 5 J/cm(2). We speculated that, at least in vitro and at our experimental conditions, UVA irradiation decreases mast cells-VEGF release and gene-expression., (© 2012 John Wiley & Sons A/S.)

IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.

Polyphenols are the abundant micronutrient in our diet and attention has been given to them for the prevention of degenerative diseases. Since over production of ROS and proinflammatory cytokine are often act as the triggers for the promotion stage of carcinogenesis by transcriptional up-regulation of nuclear factor-κB (NF-κB) and cycloxygenage-2 (COX-2). We investigated the protective effects of caffeic acid (CA) on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced oxidative and inflammatory responses, expression of NF-κB and COX-2 in mouse skin. Animals were given pre-treatment of CA at two different doses 10μmol (D1) and 20μmol (D2)/0.2ml of acetone 30min prior to each TPA (10nmol/0.2ml of acetone) application. Our results show that CA significantly inhibit the TPA induced lipid peroxidation (LPO), inflammatory responses, tumor necrosis factor alpha (TNF-α) release and also found to up regulate GSH content and the activity of different antioxidant enzymes. Further, CA was found to inhibit the TPA induced expression of NF-κB and COX-2. Thus, our results suggest that CA attenuates TPA induced tumor promotional triggers possibly by inhibition of oxidative and inflammatory responses thereby diminishing the expression of NF-κB and COX-2., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis, and its expression level strongly correlates with the severity of symptoms. However, the mechanism underlying this remains unknown. Here we report the mechanism of H1R gene up-regulation. The luciferase assay revealed the existence of two promoter regions, A and B1. Two AP-1 and one Ets-1 bound to region A, while Ku86, Ku70, and PARP-1 bound to region B1. Ku86 was responsible for DNA binding and poly(ADP-ribosyl)ated in response to phorbol-12-myristate-13-acetate stimulation, inducing its dissociation from region B1 that is crucial for promoter activity. Knockdown of Ku86 gene enhanced up-regulation of H1R gene expression. Experiments using inhibitors for MEK and PARP-1 indicate that regions A and B1 are downstream regulatory elements of the PKCδ/ERK/PARP-1 signaling pathway. Data suggest a novel mechanism for the up-regulation of H1R gene expression.

Regulation of gene expression via brain-derived neurotrophic factor (BDNF) is critical to the development of the nervous system and may well underlie cognitive performance throughout life. We now describe a mechanism by which BDNF can exert its effects on postsynaptic receptor populations that may have relevance to both the normal and diseased brain where BDNF levels either rise or fall in association with changes in excitatory neurotransmission. Increased levels of NMDA receptors (NMDARs) occur in rat cortical neurons via synthesis of new NMDA receptor 1 (NR1) subunits. The majority of synthesis is controlled by binding of cAMP response element binding protein (CREB) and early growth response factor 3 (Egr3) to the core NR1 promoter (NR1-p) region. BDNF-mediated NR1 transcription depends upon induction of the mitogen-activated protein kinase (MAPK) pathway through activation of the TrK-B receptor. Taken together with the fact that NMDAR activation stimulates BDNF synthesis, our results uncover a feed-forward gene regulatory network that may enhance excitatory neurotransmission to change neuronal behavior over time., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

CD3 is a complex of polypeptides which form part of the T cell receptor. Normal human peripheral pan T cells, express not only CD3, but the mRNA for myosin regulatory light chains MYL9, MYL12A, and MYL12B are also significantly expressed. In the Jurkat wild strain, an acute T cell leukemia cell line, CD3 on the surface and MYL9 mRNA are not expressed, while both MYL12A mRNA and MYL12B mRNA are expressed. Jurkat-I, a new clone was established by the transfection of the MYL9 gene into the Jurkat wild strain. As a result, the level of CD3 expressed on the surface of Jurkat-I cells was significantly higher than those in the Jurkat wild strain. Phorbol 12-myristate 13-acetate increased the surface CD3 levels in Jurkat wild strain cells without resulting in MYL9 gene expression, indicating that protein kinase C is partially involved in the expression of CD3 on the surface. These results suggest that surface CD3 expression proceeds through both MYL9-dependent and MYL9-independent pathways (i.e. the protein kinase C- dependent pathway) in Jurkat cells.

During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

Cigarette smoke could induce pulmonary smooth muscle cells (PASMCs) proliferation. Although our previous study had implied the involvement of protein kinase Cα (PKCα), the molecular mechanism underlying PKCα pathway in this process is still unknown. In this study, rat PASMCs were stimulated by cigarette smoke extract (CSE) or PMA (a special activator to PKCα). Two percent CSE and PMA significantly enhanced cyclin D1 expression and cells proliferation. But cyclin D1-specific siRNA successfully inhibited DNA synthesis in CSE-treated or PMA-treated cells. On the other hand, PKCα-specific siRNA significantly suppressed cyclin D1 expression in CSE-treated cells. Moreover, PKCα-specific siRNA resulted in a cell-cycle arrest in G0/G1 and decreased cells number significantly. We conclude that CSE induced rat PASMCs proliferation at least partly via PKCα-mediated cyclin D1 expression., (Copyright © 2011 Wiley-Liss, Inc.)

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.

Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells.

Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease. Although there are many reports regarding function and regulatory mechanism of TSLP in dendritic cells and/or T cells, the regulatory mechanism of TSLP in mast cells has not been fully elucidated. Here, we describe how TSLP is expressed and produced by inflammatory stimulus in mast cells. TSLP mRNA was expressed by phorbol myristate acetate (PMA) plus A23187 stimulation in HMC-1 cells and reached its peak 5h after PMA plus A23187 stimulation. The expression of TSLP mRNA was inhibited by nuclear factor (NF)-κB inhibitor. In addition, NF-κB luciferase activity was inhibited by caspase-1 inhibitor, indicating that caspase-1 is an upstream of NF-κB in mast cells. Furthermore, caspase-1 inhibitor decreased the expression of TSLP mRNA induced by PMA plus A23187. Finally, TSLP production was inhibited by both caspase-1 inhibitor and NF-κB inhibitor. These results provide proof of principle that TSLP can be expressed and produced through caspase-1 and NF-κB in mast cells and open new perspectives to pharmacologically manipulate the expression and production of TSLP by molecules acting on the caspase-1 and NF-κB pathway., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5' end or 131 bp from the 3' end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal -100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5' end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174-182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.

Sustained neuronal activity induces synaptic remodeling, in part, by altering gene expression. We have used a major climbing fiber pathway onto cerebellar Purkinje cells to investigate the effects of sustained climbing fiber-evoked glutamatergic synaptic transmission on transcription, expression and phosphorylation of proteins related to the regulation of inhibitory GABA(A) receptor function. Binocular horizontal optokinetic stimulation was used to modulate climbing fiber signals to Purkinje cells in the flocculus and nodulus of rabbits and mice. Purkinje cells in the flocculus and nodulus ipsilateral to the eye stimulated in the Posterior→Anterior direction received increased climbing fiber activity. Purkinje cells in flocculus and nodulus ipsilateral to the eye stimulated in the Anterior→Posterior direction received decreased climbing fiber activity. We identified changes in levels of gene transcripts in floccular and nodular Purkinje cells with the technique of differential display RT-PCR. Increased climbing fiber input reduced transcript levels and expression of GABA receptor-associated protein (GABARAP). Using a protein 'pull down' technique, we showed that GABARAP interacts with serine phosphorylated GABA(A)γ2, gephyrin and β-tubulin. Serine de-phosphorylation of GABA(A)γ2 reduced association of GABARAP with GABA(A)γ2. Climbing fiber activity did not influence the expression of GABA(A)γ2. Rather, it decreased its serine phosphorylation. Climbing fiber discharge decreased both expression of GABARAP and serine phosphorylation of GABA(A)γ2. Consequently, climbing fiber activity may reduce the surface expression of GABA(A) receptors in Purkinje cells rendering Purkinje cells less susceptible to interneuronal GABAergic inhibition., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Background: Among several GATA family transcription factor-associating proteins, zinc finger protein, multitype 1 (ZFPM1), at least that of murine origin, has been shown to modulate the activity of GATA-3. However, the functional role of human ZFPM1 in the immune system has not been elucidated. Therefore, we here investigated the contribution of ZFPM1 to human Th1/Th2 differentiation., Methods: The cDNA of ZFPM1 was cloned and introduced into human cord blood CD4+ T cells by a lentiviral transduction system. Then, the expression of IL-4 and IFN-γ mRNA was determined by quantitative real-time RT-PCR. The effect of ZFPM1 on the promoter activity of IL-4 and IFN-γ in Jurkat cells was also investigated., Results: Stimulation-induced expression of IL-4 and IFN-γ in human CD4+ T cells was suppressed and enhanced, respectively, by the introduction of ZFPM1. The transcriptional activity of IL-4 was also diminished by ZFPM1, whereas that of IFN-γ was not affected., Conclusion: ZFPM1 that facilitates human Th1 differentiation via the downregulation of IL-4 is a potential target for the treatment of allergic diseases., (Copyright © 2011 S. Karger AG, Basel.)