Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Cross-sectional study of a microsatellite marker in the low density lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO2 group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.

Design and synthesis of novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives as thyroid hormone receptor β (TR-β) selective ligands

Design and synthesis of a novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives are reported and their in vitro thyroid hormone receptor selectivity has been evaluated in the thyroid luciferase receptor assay. The 3-[3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)-phenylamino]-4-hydroxy-cyclobut-3-ene-1,2-dione 21 has shown selectivity towards thyroid hormone receptor β.

YB-1, a key regulator of androgen receptor signalling

Reactivation of androgen receptor signalling is one of the hallmarks of prostate cancer progression to the terminal castrate resistant stage. A better understanding of mechanisms driving this adaptive response is essential for the development of innovative intervention strategies that effectively delay or halt prostate cancer progression. The Y-box binding protein 1 (YB-1) has been found to be closely associated with prostate cancer progression. By characterising its role in the adaptive process leading to castrate resistance, we aim to promote YB-1 as a novel therapeutic target in advanced prostate cancer.

Mutational analysis of the insulin‑like growth factor 1 receptor tyrosine kinase domain in non-small cell lung cancer patients

The insulin‑like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non‑small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well‑documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single‑nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16‑21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease‑free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.

Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

Temozolomide-modulated glioma proteome: Role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR andWestern blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Novel multimeric IL-1 receptor antagonist for the treatment of rheumatoid arthritis

Protein therapeutics targeting inflammatory mediators have shown great promise for the treatment of autoimmunities such as rheumatoid arthritis (RA). However, a significant challenge in this area has been their low in vivo stability and consequently their severely compromised therapeutic efficacy. One such therapeutic molecule IL-1 receptor antagonist (IL-1ra), used in the treatment of rheumatoid arthritis, has displayed only modest efficacy in human clinical trials owing to its short biological half-life. Herein, we report a novel approach to conglomerate individual protein entities into a drug depot by incorporation of an amyloidogenic motif Lys-Phe-Phe-Glu (KFFE) thereby dramatically improving their systemic persistence and in turn their therapeutic efficacy in a mice model of autoimmune arthritis. (C) 2014 Elsevier Ltd. All rights reserved.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

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HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

Visualization by High Resolution Immunoelectron Microscopy of the Transient Receptor Potential Vanilloid-1 at Inhibitory Synapses of the Mouse Dentate Gyrus

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (similar to 60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present.

Estudo do polimorfismo Ser49Gly do gene do receptor beta adrenérgico 1 (β-adr 1) na população do estado do Rio de Janeiro, Brasil estratificada por cor da pele e ancestralidade genômica

As doenças cardiovasculares possuem a maior taxa de óbitos no mundo, e notavelmente nos últimos anos as pesquisas genéticas sobre as mesmas estão baseadas em estudos de associação, no qual o gene suspeito que esteja em maior frequência entre os pacientes passa a ser considerado um possível fator causal. Os polimorfismos genéticos que ocorrem no receptor beta-adrenérgico podem resultar em mudanças significativas na função do receptor, podendo acarretar fisiopatologias. Neste trabalho, o objetivo foi estimar a diversidade e a frequência do polimorfismo Ser49Gly do gene do receptor beta-adrenérgico 1 a partir de uma amostra de 188 indivíduos da população do Estado do Rio de Janeiro. As frequências também foram analisadas a partir da estratificação da amostra por critério fenotípico em função do padrão de cor da pele em (negros e não negros) ou ancestralidade genética em (afrodescendente e não afrodescendente), definida através da informação dos marcadores de ancestralidade Indels e SNP de cromossomo Y, para avaliar se os padrões de ancestralidade ou cor da pele são fundamentais para a diferenciação e distanciamento genético. Fragmentos de interesse foram amplificados por PCR (reação de cadeia de polimerase) com primers específicos para o marcador Ser49Gly e as reações de genotipagem foram realizadas com enzimas de restrição Eco0109I. Os valores da heterozigosidade variaram entre 0,25-0,50 e 0,20-0,41 nos grupos estratificados por ancestralidade e cor da pele, respectivamente. No que diz respeito à análise do equilíbrio de Hardy-Weinberg, não houve um desvio significativo na distribuição do marcador nas amostras gerais do Estado do Rio de Janeiro, ou mesmo nas amostras estratificadas. A distribuição dos alelos na amostra dos 188 indivíduos da população geral do Rio de Janeiro (AC_RJ) mostrou uma frequência de 80,30% e 19,70% para o alelo selvagem e mutado Ser49Gly, respectivamente. A comparação das análises sobre a distribuição das frequências alélicas para este marcador mostrou a ocorrência de diferenças significativas na distribuição das frequências alélicas entre negros e não negros e afrodescendentes e não afrodescendentes. A diferença significativa observada entre os negros e afrodescendentes, foi em menor grau de distanciamento. A informação obtida em relação à ancestralidade foi crucial para a obtenção dos dados sobre o aumento da variável mutada do polimorfismo Ser49Gly nas populações negras e afrodescendentes do Estado Rio de Janeiro. Tal evidência, em combinação com estudos clínicos podem contribuir para uma análise pormenorizada do padrão de susceptibilidade à doença em questão, em falhas do mecanismo deste receptor.