HIF-1 alpha (1772C > T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1 alpha is rapidly degraded by von Hippel-Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1 alpha (1772C > T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1 alpha (1772C > T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1 alpha (1772C > T) (rs 11549465) polymorphism.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

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.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

N-methyl-D-aspartate receptor-dependent long-term potentiation in CA1 region affects synaptic expression of glutamate receptor subunits and associated proteins in the whole hippocampus

Long term potentiation in hippocampus, evoked by high-frequency stimulation, is mediated by two major glutamate receptor subtypes, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptors. Receptor subunit compos

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Pharmacological and immunocytochemical investigation of the role of catecholamines on larval metamorphosis by beta-adrenergic-like receptor in the bivalve Meretrix meretrix

Catecholamines regulate several physiological processes in mollusks. Many pharmacological experiments have been conducted to determine the effects of adrenergic agonist and antagonist of catecholamine receptors on Meretrix meretrix metamorphosis. Results showed that adrenaline (AD) and noradrenaline (NA) had substantial effects (p < 0.05) on larval metamorphosis at concentrations ranging from 10 mu M to 100 mu M. 10 mu M beta-adrenergic receptor (AR) agonist isoproterenol showed the same inducement effect as that of NA and AD on metamorphosis, whereas the alpha-AR agonist phenylephrine had no significant effect at concentrations between 0.1 mu M and 100 mu M concentrations (p > 0.05). Furthermore, I mu M beta-AR antagonist propanolol, but not alpha-AR antagonist prazosin, depressed the larval metamorphosis induced by NA or AD. By immunocytochemistry, two cell bodies of beta-adrenergic-like receptor, C/A1, C/A2, were observed in the cerebral/apical ganglion of competent larvae. In addition, there were other immunoreactive dots near C/A1 and C/A2. The results of pharmacology and immunocytochemistry suggests that beta-adrenergic-like receptor located in the larval CNS, might play a considerable role in the larval metamorphosis of M meretrix by AD or NA. (c) 2006 Elsevier B.V. All rights reserved.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

Molecular mechanisms underpinning IFN-gamma and TNF-alpha synergy in intestinal epithelial cells

Cytokine-driven signalling shapes immune homeostasis and guides inflammatory responses mainly through induction of specific gene expression programmes both within and outside the immune cell compartment. These transcriptional outputs are often amplified via cytokine synergy, which sets a stimulatory threshold that safeguards from exacerbated inflammation and immunopathology. In this study, we investigated the molecular mechanisms underpinning synergy between two pivotal Th1 cytokines, IFN-γ and TNF-α, in human intestinal epithelial cells. These two proinflammatory mediators induce a unique state of signalling and transcriptional synergy implicated in processes such as antiviral and antitumour immunity, intestinal barrier and pancreatic β-cell dysfunction. Since its discovery more than 30 years ago, this biological phenomenon remains, however, only partially defined. Here, using a functional genomics approach including RNAi perturbation screens and small-molecule inhibitors, we identified two new regulators of IFN-γ/TNF-α-induced chemokine and antiviral gene and protein expression, a Bcl-2 protein BCL-G and a histone demethylase UTX. We also discovered that IFN-γ/TNF-α synergise to trigger a coordinated shutdown of major receptor tyrosine kinases expression in colon cancer cells. Together, these findings extend our current understanding of how IFN-γ/TNF-α synergy elicits qualitatively and quantitatively distinct outputs in the intestinal epithelium. Given the well-documented role of this synergistic state in immunopathology of various disorders, our results may help to inform the identification of high quality and biologically relevant druggable targets for diseases characterised by an IFN-γ/TNF-α high immune signature

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Bbeta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: regulation by beta- but not alpha1-adrenergic stimulation.

Pressure overload ventricular hypertrophy is accompanied by dysfunctional beta-adrenergic receptor signaling due to increased levels of the beta-adrenergic receptor kinase-1, which phosphorylates and desensitizes beta-adrenergic receptors. In this study, we examined whether increased beta-adrenergic receptor kinase 1 expression is associated with myocardial hypertrophy induced by adrenergic stimulation. With use of implanted mini-osmotic pumps, we treated mice with isoproterenol, phenylephrine, or vehicle to distinguish between alpha1- and beta-adrenergic stimulation. Both treatments resulted in cardiac hypertrophy, but only isoproterenol induced significant increases in beta-adrenergic receptor kinase-1 protein levels and activity. Similarly, in isolated adult rat cardiac myocytes, 24 hours of isoproterenol stimulation resulted in a significant 2.8-fold increase in beta-adrenergic receptor kinase-1 protein levels, whereas 24 hours of phenylephrine treatment did not alter beta-adrenergic receptor kinase-1 expression. Our results indicate that increased beta-adrenergic receptor kinase-1 is not invariably associated with myocardial hypertrophy but apparently is controlled by the state of beta-adrenergic receptor activation.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.