NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam cell formation.     

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.     

Toxoplasma gondii: siRNA can mediate the suppression of adenosine kinase expression

Adenosine kinase (AK) is one of the most important enzymes in the Toxoplasma gondii purine salvage pathway. Three siRNAs specific to the AK gene were designed in the present study. At 24h following electroporation, two of them (siRNA786 and siRNA1200) significantly reduced the mRNA level compared with mock electroporation (P <0.05). The ability to incorporate [3H]-adenosine in the parasites electroporated with 4 microM siRNA786 or 4 microM siRNA1200 was decreased to 39+/-11% and 39+/-7% of the mock electroporation, respectively. At the 48th hour of electroporation, the enzyme's activity was still significantly lower than that of mock electroporation. The data show the siRNAs transfected into cells can work efficiently to regulate gene expression in T. gondii. The application of siRNA in interrupting gene expression in T. gondii would be useful for elucidating gene function as a step toward development of anti-toxoplasmasis vaccines and therapeutic reagents.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.     

HGF and Direct Mesenchymal Stem Cells Contact Synergize to Inhibit Hepatic Stellate Cells Activation through TLR4/NF-kB Pathway

Aims

Bone marrow-derived mesenchymal stem cells (BMSCs) can reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs.

Methods

We used BMSCs directly and indirectly co-culture system with HSCs to evaluate the anti-fibrosis effect of BMSCs. Cell proliferation and activation were examined in the presence of BMSCs and HGF. c-met was knockdown in HSCs to evaluate the effect of HGF secreted by BMSCs. The TLR4 and Myeloid differentiation primary response gene 88(MyD88) mRNA levels and the NF-kB pathway activation were determined by real-time PCR and western blotting analyses. The effect of BMSCs on HSCs activation was investigated in vitro in either MyD88 silencing or overexpression in HSCs. Liver fibrosis in rats fed CCl₄ with and without BMSCs supplementation was compared. Histopathological examinations and serum biochemical tests were compared between the two groups.

Results

BMSCs remarkably inhibited the proliferation and activation of HSCs by interfering with LPS-TLR4 pathway through a cell–cell contact mode that was partially mediated by HGF secretion. The NF-kB pathway is involved in HSCs activation inhibition by BMSCs. MyD88 over expression reduced the BMSC inhibition of NF-kB luciferase activation. BMSCs protected liver fibrosis in vivo.

Conclusion

BMSCs modulate HSCs in vitro via TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secreting HGF. BMSCs have therapeutic effects on cirrhosis rats. Our results provide new insights into the treatment of hepatic fibrosis with BMSCs.     

Caesalpinia sappan extract inhibits IL1β-mediated overexpression of matrix metalloproteinases in human chondrocytes

Exacerbated production of matrix metalloproteinases (MMPs) is a key event in the progression of osteoarthritis (OA) and represents a promising target for the management of OA with nutraceuticals. In this study, we sought to determine the MMP-inhibitory activity of an ethanolic Caesalpinia sappan extract (CSE) in human OA chondrocytes. Thus, human articular chondrocytes isolated from OA cartilage and SW1353 chondrocytes were stimulated with Interleukin-1beta (IL1β), without or with pretreatment with CSE. Following viability assays, the production of MMP-2 and MMP-13 was assessed using ELISA, whereas mRNA levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13 and TIMP-1, TIMP-2, TIMP-3 were quantified using RT-qPCR assays. Chondrocytes were co-transfected with a MMP-13 luciferase reporter construct and NF-kB p50 and p65 expression vectors in the presence or absence of CSE. In addition, the direct effect of CSE on the proteolytic activities of MMP-2 was evaluated using gelatin zymography. We found that CSE significantly suppressed IL1β-mediated upregulation of MMP-13 mRNA and protein levels via abrogation of the NF-kB(p65/p50)-driven MMP-13 promoter activation. We further observed that the levels of IL1β-induced MMP-1, MMP-3, MMP-7, and MMP-9 mRNA, but not TIMP mRNA levels, were down-regulated in chondrocytes in response to CSE. Zymographic results suggested that CSE did not directly interfere with the proteolytic activity of MMP-2. In summary, this study provides evidence for the MMP-inhibitory potential of CSE or CSE-derived compounds in human OA chondrocytes. The data indicate that the mechanism of this inhibition might, at least in part, involve targeting of NF-kB-mediated promoter activation.     

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P < 0.01) change in expression levels were screened according to the screening standard (signal log ratio ≥1 or ≤?1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335 ± 0.0116 72 h after transfection. This value was significantly (P < 0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P < 0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.     

The Arabidopsis thaliana FASCICLIN LIKE ARABINOGALACTAN PROTEIN 4 gene acts synergistically with abscisic acid signalling to control root growth

Background and Aims

The putative FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 4 (At-FLA4) locus of Arabidopsis thaliana has previously been shown to be required for the normal growth of wild-type roots in response to moderately elevated salinity. However, the genetic and physiological pathway that connects At-FLA4 and normal root growth remains to be elucidated.

Methods

The radial swelling phenotype of At-fla4 was modulated with growth regulators and their inhibitors. The relationship of At-FLA4 to abscisic acid (ABA) signalling was analysed by probing marker gene expression and the observation of the At-fla4 phenotype in combination with ABA signalling mutants.

Key Results

Application of ABA suppresses the non-redundant role of At-FLA4 in the salt response. At-FLA4 positively regulates the response to low ABA concentration in roots and is required for the normal expression of ABA- and abiotic stress-induced genes. The At-fla4 phenotype is enhanced in the At-abi4 background, while two genetic suppressors of ABA-induced gene expression are required for salt oversensitivity of At-fla4. Salt oversensitivity in At-fla4 is suppressed by the CYP707A inhibitor abscinazole E2B, and salt oversensitivity in At-fla4 roots is phenocopied by chemical inhibition of ABA biosynthesis.

Conclusions

The predicted lipid-anchored glycoprotein At-FLA4 positively regulates cell wall biosynthesis and root growth by modulating ABA signalling.     

The receptor guanylate cyclase Gyc76C and a peptide ligand, NPLP1-VQQ, modulate the innate immune IMD pathway in response to salt stress

Receptorguanylate cyclases (rGCs) modulate diverse physiological processes including mammalian cardiovascular function and insect eclosion. The Drosophila genome encodes several receptor and receptor-like GCs, but no ligand for any Drosophila rGC has yet been identified. By screening peptide libraries in Drosophila S2 cells, the Drosophila peptide NPLP1-VQQ (NLGALKSSPVHGVQQ) was shown to be a ligand for the rGC, Gyc76C (CG42636, previously CG8742, l(3)76BDl, DrGC-1). In the adult fly, expression of Gyc76C is highest in immune and stress-sensing epithelial tissues, including Malpighian tubules and midgut; and NPLP1-VQQ stimulates fluid transport and increases cGMP content in tubules. cGMP signaling is known to modulate the activity of the IMD innate immune pathway in tubules via activation and nuclear translocation of the NF-kB orthologue, Relish, resulting in increased anti-microbial peptide (AMP) gene expression; and so NPLP1-VQQ might act in immune/stress responses. Indeed, NPLP1-VQQ induces nuclear translocation of Relish in intact tubules and increases expression of the anti-microbial peptide gene, diptericin. Targeted Gyc76C RNAi to tubule principal cells inhibited both NPLP1-VQQ-induced Relish translocation and diptericin expression. Relish translocation and increased AMP gene expression also occurs in tubules in response to dietary salt stress. Gyc76C also modulates organismal survival to salt stress - ablation of Gyc76C expression in only tubule principal cells prevents Relish translocation, reduces diptericin expression, and reduces organismal survival in response to salt stress. Thus, the principal-cell localized NPLP1-VQQ/Gyc76C cGMP pathway acts to signal environmental (salt) stress to the whole organism.     

Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.     

Methylated chrysin induces co-ordinated attenuation of the canonical Wnt and NF-kB signaling pathway and upregulates apoptotic gene expression in the early hepatocarcinogenesis rat model

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer^32/36 in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr³⁹³ and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β^Ser9. Dietary DMF (100 mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.     

Genetic differ in TLR4 gene polymorphisms and expression involved in Salmonella natural and artificial infection respectively in Chinese native chicken breeds

Salmonella enteritidis (SE) is a foodborne pathogen that negatively affects both animal and human health. Genetic variations in response to pathogenic SE colonization or to SE vaccination were measured in chicken resource populations. Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. In this study, TLR4 was investigated the association of TLR4 gene polymorphisms with Salmonella natural infection situation of birds from two distinct Chinese genetic breeds. One SNP G1894C in the second intron of chicken TLR4 (chTLR4) was scanned in the two hens breed, which showed significant association with Salmonella natural infection situation (P < 0.05). Genetic variations in response to pathogenic SE colonization also existed in distinct Chinese chicken resource population. In this study, mRNA expression of TLR4 was detected to investigate the association with the effect of artificial SE challenge in heterophil granulocytes and spleen of chicks from two distinct Chinese genetic breeds at 1, 3 and 10 day post-infection during the acute infection period. It clearly showed that young chicks’ response to SE infection was regulated by TLR4 mRNA expression. The results suggest that genetics, time, gender, and interactions among these factors, play important roles in TLR4 mRNA basic values and copies modulation of SE mediated immune response in distinct Chinese chickens.