Expression Of Downstream Signaling Molecules Research Articles

Unraveling the unphosphorylated STAT3-unphosphorylated NF-κB pathway in loss of function STAT3 Hyper IgE syndrome.

Patients with loss of function signal transducer and activator of transcription 3-related Hyper IgE Syndrome (LOF STAT3 HIES) present with recurrent staphylococcal skin and pulmonary infections along with the elevated serum IgE levels, eczematous rashes, and skeletal and facial abnormalities. Defective STAT3 signaling results in reduced Th17 cells and an impaired IL-17/IL-22 response primarily due to a compromised canonical Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that involves STAT3 phosphorylation, dimerization, nuclear translocation, and gene transcription. The non-canonical pathway involving unphosphorylated STAT3 and its role in disease pathogenesis, however, is unexplored in HIES. This study aims to elucidate the role of unphosphorylated STAT3-unphosphorylated NF-κB (uSTAT3-uNF-κB) activation pathway in LOF STAT3 HIES patients. The mRNA expression of downstream molecules of unphosphorylated STAT3-unphosphorylated NF-κB pathway was studied in five LOF STAT3 HIES patients and transfected STAT3 mutants post-IL-6 stimulation. Immunoprecipitation assays were performed to assess the binding of STAT3 and NF-κB to RANTES promoter. A reduced expression of the downstream signaling molecules of the uSTAT3-uNF-κB complex pathway, viz., RANTES, STAT3, IL-6, IL-8, ICAM1, IL-8, ZFP36L2, CSF1, MRAS, and SOCS3, in LOF STAT3 HIES patients as well as the different STAT3 mutant plasmids was observed. Immunoprecipitation studies showed a reduced interaction of STAT3 and NF-κB to RANTES in HIES patients. The reduced expression of downstream signaling molecules, specially RANTES and STAT3, confirmed the impaired uSTAT3-uNF-κB pathway in STAT3 LOF HIES. Decreased levels of RANTES and STAT3 could be a significant component in the disease pathogenesis of Hyper IgE Syndrome.

Placenta-specific 8 facilitates the infection of duck hepatitis A virus type 1 by inhibiting the TLR7 MyD88-dependent signaling pathway

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication.

Mycobacterium tuberculosis dormancy regulon proteins Rv2627c and Rv2628 as Toll like receptor agonist and as potential adjuvant

During latency, DosR proteins of Mycobacterium tuberculosis (M.tb) get activated and help the bacterium to remain dormant. We have shown earlier that 2 such proteins Rv2627c and Rv2628 are immunogenic and induce a TH1 kind of immune response. In this study, through in-vitro experiments we have confirmed that Rv2627c and Rv2628 proteins act as protein Toll-Like Receptor (TLR) agonist-adjuvant. Rv2627c and Rv2628 stimulated THP-1 macrophages showed an increased expression of TLR2, TLR4 and co-stimulatory molecules CD40, CD80, CD86 and antigen presenting molecule HLA-DR. Further studies also found enhanced expression of downstream signaling molecules of TLR activation like MyD88, NF-κB-p65 and pro-inflammatory cytokines. Inhibition studies using TLR blocking antibodies decreased the expression of co-stimulatory molecules, MyD88, NF-κB-p65, and pro-inflammatory cytokines. Rv2627c and Rv2628 stimulation of HEK-TLR2 reporter cell line confirmed the interaction of these proteins with TLR2. Moreover, molecular docking and simulations of Rv2627c and Rv2628 proteins with TLR2 and TLR4 showed stable interactions. The adjuvant activity of Rv2628 was further validated by a protein adjuvanted with pre-clinically validated peptides as multi-epitope vaccine construct which showed good binding with TLR2 and TLR4 and activate dendritic cells and induce sustained pro-inflammatory cytokine response by C-ImmSim analysis. We propose that our vaccine construct will produce a better immune response than BCG and can be taken up as a post-exposure therapeutic subunit vaccine along with standard TB therapy. We also anticipate that our construct can be taken up as a protein adjuvant with other vaccine candidates as these can activate macrophages through TLR signaling.

Semi-Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity.

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC50 value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature.

PurposeROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of ROR2 in children with short stature and to identify the underlying molecular mechanisms.MethodsWe retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of ROR2. We subsequently examined the changes in protein expression and subcellular location in ROR2 caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a–ROR2 pathway was also examined.ResultsWe identified 12 mutations in ROR2 in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments in vitro. The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2.ConclusionWe expanded the mutational spectrum of ROR2 in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in ROR2 might affect the expression of downstream Wnt5a–ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.

Inhibition of Macrophage Migration Inhibitory Factor Protects against Inflammation through a Toll-like Receptor-Related Pathway after Diffuse Axonal Injury in Rats.

Objective We have previously demonstrated that inflammation induced by toll-like receptors (TLRs) 2/4 exert cerebral deleterious effects after diffuse axonal injury (DAI); however, the underlying mechanisms are not fully understood. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine involved in inflammatory responses. The purpose of this study was to investigate the role of MIF in inflammation induced by TLRs in the cortices of DAI rats. Methods The rat DAI model was established by head rotational acceleration and confirmed by β-APP, HE, and silver staining. MIF protein expression at 3 h, 6 h, 12 h, 1 d, and 3 d after DAI was measured by western blot. The localization of MIF was measured by immunofluorescence. MIF antagonist ISO-1 was intracerebroventricularly injected to inhibit MIF. Neuronal and axonal injury and glial responses were assessed by TUNEL, immunohistochemistry, and TEM. Expression of TLR2, TLR4, ERK, phospho-ERK, NF-κB, and phospho-NF-κB was examined by western blot. The level of IL-1β, IL-6, and TNF-α was measured by ELISA. Results MIF expression was significantly increased, peaking at 1 day after DAI, and MIF was mainly localized in microglial cells and neurons. ISO-1 suppressed neuronal apoptosis, axonal injury, and glial responses and decreased the expression of downstream signaling molecules related to TLR2/4, including ERK, phospho-ERK, NF-κB, phospho-NF-κB, IL-1β, IL-6, and TNF-α. Conclusion MIF was involved in the neuronal and axonal damage through a TLR-related pathway following DAI.

Neutralization of TNF-α and IL-1β Regulates CXCL8 Production through CXCL8/CXCR1 Axis in Macrophages during Staphylococcus aureus Infection

ABSTRACT Anti-cytokine therapy is widely acknowledged as an anti-inflammatory technique to treat varied infectious diseases. TNF-α and IL-1β are major cytokines that regulate every aspect of the inflammatory process. However, the effects of single or dual cytokine neutralization on S. aureus mediated CXCL8 secretion and CXCR1 expression in murine peritoneal macrophages remained noninvestigated. Thus we aimed to explore the effects of kinetic-dose dependent neutralization of TNF-α and IL-1β using specific anti-cytokine antibodies and its influential impact on the CXCL8/CXCR1 axis at different stages of S. aureus (30, 60, and 90 min) infection. The murine peritoneal macrophages were isolated and infected with viable S. aureus followed by subsequent addition of anti-TNF-α and anti-IL-1β into the medium. The treated cells were centrifuged and lysate and supernatant collected for various experiments. The ROS generation was measured and cytokine production was estimated by ELISA. The expression of TNFR1, IL-1R, CXCR1, signaling molecules (NF-κB and JNK) were evaluated by Western blot. The role of single or dual cytokine neutralization on intracellular bacterial phagocytosis had also been analyzed by confocal microscopy. Dual cytokine neutralization significantly suppressed ROS, cytokines, CXCL8 secretion, and intracellular bacterial count compared to single cytokine neutralization and it was more apparent at 90 min post S. aureus infection. There was a drastic reduction in TNFR1, IL-1R, and CXCR1 expression on macrophage surface due to reduced expression of downstream signaling molecules, NF-κB and JNK. Hence dual cytokine neutralization was more effectual compared to single cytokine neutralization in the downregulation of S. aureus induced CXCR1 expression.

Discovery of (4-bromophenyl)(3-hydroxy-4-methoxyphenyl)methanone through upregulating hTERT induces cell apoptosis and ERS

Dominant-negative mutants of telomerase hTERT were demonstrated to have selective effects in tumor cells. However, no any effective and highly selective hTERT inhibitor has been developed so far. We focused on developing new hTERT modulators and synthesized a small molecular compound, named (4-bromophenyl)(3-hydroxy-4-methoxyphenyl)methanone. Our in vitro studies found that title compound showed high inhibitory activity against telomerase, had high antiproliferative capacity on SMMC-7721 cells with IC50 value 88 nm, and had no obvious toxic effect on human normal hepatocyte cells with IC50 value 10 μM. Our in vivo studies showed that this compound significantly inhibited tumor growth in xenograft tumor models. The further molecular mechanisms of title compound inhibition SMMC-7721 cell proliferation by modulating hTERT were explored; the results showed that endoplasmic reticulum stress (ERS) through ER over response (EOR) activates the expression of hTERT, and then induces ERS, which is believed to be intricately associated with oxidative stress and mitochondrial dysfunction, resulting in apoptotic cell death, thereby modulating the expression of downstream signaling molecules including CHOP (CAAT/enhancer-binding protein homologous protein)) and mitochondrion pathway of apoptosis, leading to inhibition of cell proliferation.

Abstract 2636: Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal form of primary breast cancer. Inflammatory signaling pathways are active in IBC, but the function of the immune response remains elusive. ErbB receptors play a role in IBC. ERBB2 is amplified in 50% of IBCs, and ERBB3 is also mutated in IBC. Currently, Lapatinib, a dual ErbB inhibitor, is used in IBC patients with ERBB2 amplification. Although immune cell inflammatory signaling may promote IBC tumor growth and metastasis, the presence of cytotoxic tumor-associated lymphocytes has also been associated with a more favorable breast cancer prognosis. The contradictory nature of the immune data highlights the need to elucidate the relationship between the immune response and subsequent treatments. Given the active IBC immune component and the recent clinical benefit of immune checkpoint inhibitors in cancer treatment, IBC’s may represent a clinically unique breast cancer population that may benefit from immunomodulating agents. Interestingly, Myc is a downstream effector of ErbB signaling, and Myc is thought to regulate the expression of immune checkpoint proteins CD47 and PD-L1. The presence of PD-L1-positive IBC immune infiltrate suggests that IBC’s may benefit from therapies that disrupt PD-L1 signaling together with ErbB inhibitors. METHODS: IHC was used to examine immune checkpoint signaling and characterize the tumor-immune infiltrate. Tumor tissues were also characterized using an RNA-seq panel that examined the expression of 377 immune-related genes. Cell lines (BT474, SKR3, AU565, and SUM225) were treated with Lapatinib and Neratinib to examine the relationship between growth factor receptors and downstream immune signaling pathways. RESULTS: RNA-seq revealed the expression of specific immunosuppressive signaling pathways in tumors. Treatment of breast cancer cells with ErbB inhibitors resulted in a decrease in the levels of PD-L1. Treatment with Lapatinib and Neratinib diminished PD-L1 in all cell lines. Levels of Phospho-Stat3 decreased in BT474 and Sum225 but not in SKBR3 and AU565, implying that PD-L1 is regulated by another mechanism (ERK-MYC). CONCLUSION: Our results provide mechanistic insight into ErbB receptor activation and the expression of downstream signaling molecules (Stat3, Myc, PD-L1, and others). In addition, our unique RNA-seq immune signature reveals the expression of several genes that may serve as biomarkers of inhibitory immune signaling pathways. Our laboratory is currently examining the correlation between PD-L1 expression and the activation of ErbB2 in model systems and clinical trials using ErbB inhibitors in combination with PD-L1 inhibitors. Our immune panel gene signature may serve as a useful diagnostic test that, in conjunction with traditional ErbB testing, can identify patients that will benefit from combination therapy of an ErbB inhibitor and an immune checkpoint inhibitor. Note: This abstract was not presented at the meeting. Citation Format: Christopher A. Hamm, Sumin Zhao, Cesar A. Santa-Maria, Massimo Cristofanilli, Neil L. Spector, Sarah Bacus. Targeted therapies to ERBB receptors downregulate expression of PD-L1: implications in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2017-2636

Resistin increases the expression of NOD2 in mouse monocytes.

Previous studies have indicated that resistin, a type of adipokine, contributes to the development of insulin resistance and type 2 diabetes mellitus, and mediates inflammatory reactions. However, a specific receptor for resistin has not yet been identified. In this study, the relationship between resistin and nucleotide-binding oligomerization domain-like receptors, as well as resistin signal transduction, was examined through transfection, quantitative polymerase chain reaction, western blot analysis and ELISA. The mRNA expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a key immune receptor related to insulin resistance, was significantly increased by resistin treatment at concentrations of 100, 150 and 200 ng/ml (P<0.05, P<0.01 and P<0.01, respectively). The mRNA expression of downstream signaling molecules in the NOD2 signaling pathway, receptor-interacting serine/threonine-protein kinase 2 (RIP2; P<0.01 at 6, 12 and 24 h) and inhibitor of NF-κB kinase subunit beta (P<0.01 at 3, 6, 12 and 24 h) were significantly increased by resistin treatment compared with the control. The mRNA expression of key proinflammatory cytokines, tumor necrosis factor α, IL (interleukin)-6 and IL-1β, were also significantly increased by resistin treatment compared with the control (P<0.01). NOD2 knockdown by small interfering RNA (siRNA) significantly decreased the expression of NOD2 and RIP2 (P<0.01), and there was no significant increase in the levels of cytokines, as compared with treatment with control siRNA. These findings indicate that the inflammatory reaction induced by resistin involves the NOD2-nuclear factor (NF)-κB signaling pathway. The inhibition of NF-κB significantly decreased the secretion of key inflammatory cytokines (P<0.01), suggesting that NF-κB signaling mechanisms are essential to the resistin-induced inflammatory response.

Novel dihydropyrazole-chromen: Design and modulates hTERT inhibition proliferation of MGC-803

Dominant-negative mutant of telomerase hTERT was demonstrated to show selective anticancer effects in tumor cells. But, an effective hTERT inhibitor with high selectivity has not been developed so far. Focused on hTERT, a novel dihydropyrazole-chromen (13k) controlling hTERT was designed. Title compound 13k occupied high antiproliferative activity against MGC-803 cells with IC50 value 1.41 μM, but it manifested obvious un-toxic effect on human normal gastric mucosa cells with the IC50 2.3 mM. Treated with compound 13k, the further inhibition mechanisms by modulating hTERT was explored, the results showed that expression of hTERT was clearly modulated, and then β-catenin activation was decreased, thereby the expression of downstream signaling molecules including c-myc and cyclin D1 was modulated, leading to inhibition MGC-803 cells proliferation.

Prevalence, Clinicopathologic Characteristics, and Molecular Associations of EGFR Exon 20 Insertion Mutations in East Asian Patients with Lung Adenocarcinoma

To define the prevalence, clinicopathologic characteristics and molecular associations of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in East Asian lung adenocarcinoma patients. A total of 1,086 lung adenocarcinomas were sequenced for EGFR mutations. EGFR and HER2 copy number variations; total and phosphorylated (p) protein expression of ErbB family members including EGFR, HER2, and HER3; phosphorylated protein expression of downstream signaling molecules including Akt and Erk; and clinicopathologic features in lung adenocarcinomas with EGFR exon 20 insertion mutations were all investigated. EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all the EGFR mutations. Compared to those with classic activating EGFR mutations, lung adenocarcinomas with exon 20 insertion mutations were characterized by significantly younger age at diagnosis (P = 0.032 for exon 20 insertions vs. L858R) and shorter relapse-free survival [P = 0.045 for exon 20 insertions versus (vs) exon 19 deletions]. Molecularly, samples harboring exon 20 insertion mutations had lower expression of phosphorylated (p)-EGFR (P < 0.001) and HER3 (P = 0.016). In addition, higher expression of p-Akt (P = 0.007) and lower expression of p-Erk (P = 0.009) were observed in tumors with exon 20 insertion mutations. Lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a substantial proportion. This subset showed distinct clinicopathologic features, less dependence on EGFR molecularly, and different pathway activation patterns compared to those with classic EGFR activating mutations.

Discovery of the novel mTOR inhibitor and its antitumor activities in vitro and in vivo.

The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examination. Compound [(E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-yl)- 3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01] was then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non-small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor-bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor.

High‐expression EGFR/cell membrane chromatography‐online‐high‐performance liquid chromatography/mass spectrometry: rapid screening of EGFR antagonists from Semen Strychni

Traditional methods for screening active compounds from complex system such as traditional Chinese medicines are relatively cumbersome and time-consuming. In order to improve this situation, we established an online analytical method for screening, separation and identification EGFR antagonists from traditional Chinese medicines, which is described in this study. Cells with high EGFR expression levels were used to prepare the cell membrane stationary phase for the EGFR cell membrane chromatography model with the purpose of screening active compounds. Separation of the retention fractions was achieved by the high-performance liquid chromatography, and identification was conducted via electrospray ionization quadrupole mass spectrometry. The inhibitory effects of active compounds on EGFR cell growth were also demonstrated in vitro. The screening results showed that vauquline and strychnine from Semen Strychni could be active components acting on EGFR similarly to gefitinib as a control drug. Results from biological trials showed that vauquline and strychnine inhibited cell proliferation of HEK293/EGFR cells, and inhibited Erk phosphorylation, and can effectively reduce expression of downstream signaling molecules. This EGFR cell membrane chromatography-online-high-performance liquid chromatography/tandem mass spectrometry method can be applied for rapid screening, separation and identification of EGFR antagonists from traditional Chinese medicines and should be useful for drug discovery with natural medicinal herbs.

Adiponectin Resistance in Heart Failure and the Emerging Pattern of Metabolic Failure in Chronic Heart Failure

In recent years, an interesting shift in our understanding of chronic heart failure (CHF) pathophysiology may be observed. Beyond the traditional concepts of hemodynamic failure and neuroendocrine activation, it is increasingly appreciated that CHF is a much more complex and truly systemic disease including the peripheral organs and whole body regulatory processes as well. Novel concepts such as the systemic inflammatory immune activation and the skeletal muscle hypothesis have emerged.1 These concepts recognize the significant contribution of peripheral changes to symptomatic status, disease progression, and outcome in CHF. Article see p 185 A further novel facet in heart failure pathophysiology has emerged recently because impaired regulation of systemic metabolic balance is increasingly in the focus of clinical research. The emerging picture suggests a complex but characteristic pattern of metabolic pathways that are imbalanced, attenuated, or abnormally activated. Hormonal imbalances have been previously observed as common features in CHF, such as insulin resistance2 and growth hormone resistance3 that contribute to both morbidity and mortality of patients. The metabolic interaction of neuroendocrine activation and immune activation such as from cytokines and oxygen radical accumulation add to the complexity of these interrelated processes. In general, the findings repeat, again, the classical concept of short-term beneficial adaptive responses on acute injury or disease may eventually turn into harmful maladaptive signals on prolonged and chronic activation. The overall clinical effects that may be observed from impaired energy metabolic efficacy contribute to impaired exercise capacity, muscle fatigue, and early exhaustion—key symptoms in heart …

Differential expression of Toll-like receptors in murine peritoneal macrophages in vitro on treatment with cisplatin

In the present study, we have investigated the differential expression of Toll-like receptors [(TLRs) 1-9] in murine peritoneal macrophages in vitro, on treatment with cis-diaminedichloroplatinum (II) (cisplatin). It is demonstrated that cisplatin induces the expression of TLRs and is a potent activator of Toll-signaling pathway. The enhanced expression of TLR2, -3, -4, -5, -6, -7, -8 and -9 is observed at different time intervals after 5 microg ml(-1) cisplatin treatment. The expression of downstream signaling molecules of TLR-signaling pathway--myeloid differentiation factor 88, IRAK1, tumor necrosis factor receptor-associated factor 6 and transcription factors IRF3 and nuclear factor-kappaB (NF-kappaB)--has also been investigated. The expression of TLR2, -3, -4 and -9 was down-regulated in cisplatin-treated macrophages in the presence of inhibitors of mitogen-activated protein kinases and NF-kappaB pathways, suggesting a role of these pathways in cisplatin-induced TLR expression. It is also observed that pre-treatment of macrophages with cisplatin and subsequent incubation with TLR ligands significantly enhanced the production of pro-inflammatory cytokines (tumor necrosis factor-alpha, IFN-gamma, IL-1beta and IL-12) and iNOS expression in macrophages. The data suggest that treatment of macrophages with cisplatin renders them more susceptible to subsequent induction of pro-inflammatory cytokines and iNOS expression by different TLR ligands. It is proposed that the pharmacological reagents like cisplatin can be used to manipulate the innate immune responses, which may be effectively used for the development of novel therapeutic approaches.

Expression of Toll-like Receptors (TLRs) and Nucleotide-binding Oligomerization Domain (NODs) in murine peritoneal macrophages on in vitro treatment with Thymosin α 1

Thymosin α 1 (Tα1) treatment of murine peritoneal macrophages in vitro induces the expression of TLRs and Nod2 proteins. Enhanced expressions of TLR2, -4, -5, -6, -7, -8, -9 and Nod2 were observed by RT-PCR and western blotting. Expression of downstream signaling molecules - MyD88, IRAK1, TRAF6, IRF3 were also up regulated on treatment with Tα1. It was also observed that pretreatment of macrophages with pharmacological inhibitors strongly down regulated the Tα1 induced expression of TLR2,-4,-9. Macrophages pretreated with Tα1 and then incubated with ligands for TLRs - Zymosan A, LPS and CpG DNA, showed significantly enhanced production of proinflammatory cytokines, than the macrophages treated with ligands alone. This suggests that pretreatment of macrophages with Tα1 makes them functionally more responsive to further challenge with TLR ligands.

UPREGULATION OF ANGIOTENSIN AT1 RECEPTOR AND INTRACELLULAR KINASE GENE EXPRESSION IN HYPERTENSIVE RATS

1. Activation of angiotensin II AT1 receptors (AT1R) stimulates catecholamine systems within both central and peripheral tissues that are associated with blood pressure control. In the present study, we sought to determine whether the hypertensive phenotype of the spontaneously hypertensive rat (SHR) is associated with changes in AT1R gene expression and whether gene expression of downstream signalling molecules was coupled to catecholamine gene expression, both in key brainstem nuclei and in peripheral sites implicated in cardiovascular control. 2. Gene expression levels of AT1R, extracellular signal-regulated kinase (ERK) 1 and 2 and phosphatidylinositol 3-kinase (PI3-K) were quantified in Wistar-Kyoto (WKY) rats and SHR. Messenger RNA expression levels were quantified using real time reverse transcription-polymerase chain reaction. In addition, we investigated whether there was a relationship between gene expression and systolic blood pressure. 3. The gene expression levels of AT1R, ERK2 and PI3-K were significantly higher in the paraventricular nucleus of the hypothalamus (4.12-, 1.40- and 1.38-fold, respectively), rostral ventrolateral medulla (2.71-, 1.33- and 2.73-fold, respectively), spinal cord (30.5-, 2.72- and 1.53-fold, respectively), adrenal medulla (1.68-, 1.55- and 1.76-fold, respectively) and coeliac ganglion (1.39-, 1.35- and 1.12-fold, respectively) in SHR compared with WKY rats. There was no significant difference in the level of ERK1 gene expression between the two strains. The gene expression levels of AT1R and ERK2 were positively correlated with blood pressure in all central nervous tissues investigated in the SHR, but not in WKY rats. Gene expression levels of the AT1R in the coeliac ganglion and adrenal medulla were also positively correlated with increased systolic blood pressure. 4. The present data suggest that a defect in AT1R expression (that may further alter downstream signalling pathways) in the SHR may be responsible, at least in part, for the hypertensive phenotype.

Suppression subtractive hybridization and microarray identification of estrogen-regulated hypothalamic genes.

The gonadal steroid estrogen is a pleiotropic hormone that has multiple effects on numerous cellular functions. One of estrogen's major targets is the brain, where the steroid not only affects growth, differentiation, and survival of neurons, but also regulates cell excitability. Because estrogen modulates multiple, overlapping signaling pathways, it has been difficult to scrutinize the transcriptional activity of the steroid. Therefore, we still lack a global picture of how different genes interact and are regulated by estrogen. Herein we report the use of suppression subtractive hybridization followed by custom microarray analysis of thousands of genes that are differentially expressed during the negative feedback phase of the female reproductive cycle. We have found a number of key transcripts that are regulated by estrogen and contribute to the alteration in synaptic transmission and hence excitability of hypothalamic neurons (e.g., GABA neurons). These include gec-1, GABA(B)R2, PI3 kinase subunit p55gamma, and a number of proteins containing pleckstrin homology domains that are critical for plasma membrane targeting. Studies are underway to refine our analysis to individual nuclei and individual cells. However, what has emerged from this highly sensitive microarray analysis is that estrogen affects neuronal plasticity in hypothalamic neurons not only by transcription of new membrane proteins (e.g., receptors and channels), but also by altering expression of downstream signaling molecules and proteins involved in neurosecretory pathways.

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Burkitt's lymphoma cell lines.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of TRAIL receptors on malignant cells. Here we have characterized the TRAIL sensitivity of a panel of Burkitt's lymphoma (BL) cell lines. Overall, 5/12 BL cell lines and 1/2 lymphoblastoid cell lines were sensitive to TRAIL-induced apoptosis, although only one BL cell line approached the sensitivity of Jurkat cells, a widely used model for TRAIL-induced apoptosis. Whereas, 4/5 of the Epstein-Barr virus (EBV)-negative cell lines were TRAIL sensitive, only 1/7 EBV-positive BL cell lines were TRAIL sensitive. However, isogenic BL cell lines with different EBV status were not differently sensitive to TRAIL, indicating that EBV is not a major determinant of TRAIL sensitivity. All cell lines expressed the death receptor (DR)5 TRAIL receptor, whereas expression of DR4 was more variable. Differences in the expression of downstream signalling molecules [Fas-associated death domain protein (FADD), caspase 8] and inhibitors [decoy receptor 1 (DcR1), cellular FLICE-like inhibitory protein (c-FLIP)] did not correlate with TRAIL sensitivity. Therefore, a subset of BL cell lines are sensitive to TRAIL-induced apoptosis, however, the molecular mechanism that determines responsiveness remains to be identified.