Beta2 Adrenergic Receptor (ADRβ2) Haplotype Pair (2/4) Is Associated with Severe Asthma

Background

β2 adrenergic receptor (ADRβ2) polymorphisms including ADRβ2+46G>A have been reported to cause adverse outcomes in mild asthmatics. The extent to which ADRβ2 polymorphisms and in particular their haplotypes contribute to severe asthma is unknown.

Objective

To determine the association of ADRβ2 polymorphisms and haplotypes with asthma severity.

Methods

Caucasians (n = 2979) were genotyped for 11 ADRβ2 polymorphisms. The cohort (mean age 39.6, 60% female) included 2296 non-asthmatics, 386 mild asthmatics, 172 moderate asthmatics and 125 severe asthmatics. Haplotype frequency and haplotype pair for each subject was determined using the PHASE algorithm.

Results

The three asthmatic cohorts were comparable in age and gender but were distinguishable from each other in terms of symptoms, spirometry, medication use and health care utilisation (p <0.001). None of the polymorphisms showed a genotypic or allelic association with asthma diagnosis or severity. Nine haplotypes were identified and no association was found with asthma diagnosis or severity per se. Haplotype pair 2/4 was associated with asthma severity (Trend Test, OR 1.42, p = 0.0008) but not with asthma per se. Prevalence of haplotype pair 2/2 appeared to decrease with asthma severity (Trend Test, OR 0.78, p = 0.067). Two new haplotypes were identified, occurring exclusively in asthmatics at a frequency of ≥ 1%. In addition, a positive association between carriage of ADRβ2 +523*C and increased risk of atopy was discovered.

Conclusions

ADRβ2 haplotype pair 2/4 is associated with severe asthma and is consistent with findings of poor bronchodilator response in mild asthmatics who are also haplotype 2/4.     

Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α₁-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α₁-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α₁-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca²⁺ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α₁-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α₁-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.     

Xenobiotic-Induced Hepatocyte Proliferation Associated with Constitutive Active/Androstane Receptor (CAR) or Peroxisome Proliferator-Activated Receptor α (PPARα) Is Enhanced by Pregnane X Receptor (PXR) Activation in Mice

Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.     

Toll-like Receptor 2 (TLR2), Transforming Growth Factor-β, Hyaluronan (HA), and Receptor for HA-mediated Motility (RHAMM) Are Required for Surfactant Protein A-stimulated Macrophage Chemotaxis

The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA- mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2^−/− mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44^−/− mice had similar responses to SPA, whereas those from RHAMM^−/− mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.     

Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr⁹²¹, Tyr⁹³⁰, and Tyr⁹⁶⁰, has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr⁹²¹ and Tyr⁹³⁰ enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.     

迷失的世界(17)

正~~     

T-cell Immunoglobulin and ITIM Domain (TIGIT) Receptor/Poliovirus Receptor (PVR) Ligand Engagement Suppresses Interferon-γ Production of Natural Killer Cells via β-Arrestin 2-mediated Negative Signaling

Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8⁺ T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor β-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.     

A QM protein–ligand investigation of antipsychotic drugs with the dopamine D2 Receptor (D2R)

The dopamine D2 Receptor (D2R) is a member of the G-Protein-Coupled Receptor family and plays a critical role in neurotransmission activities in the human brain. Dysfunction in dopamine receptor signaling may lead to mental health illnesses such as schizophrenia and Parkinson’s disease. D2R is the target protein of the commonly used antipsychotic drugs such as risperidone, clozapine, aripiprazole, olanzapine, ziprasidone, and quetiapine. Due to their significant side effects and non-selective profiles, the discovery of novel drugs has become a challenge for researchers working in this field. Recently, our group has focused on the interactions of these drug molecules in the active site of the D2R using different in silico approaches. We here compare the performances of different approaches in estimating the drug binding affinities using quantum chemical approaches. Conformations of drug molecules (ligands) at the binding site of the D2R taken from the preliminary docking studies and molecular dynamics simulations were used to generate protein–ligand interaction models. In a first approach, the BSSE-corrected interaction energies of the ligands with the most critical amino acid Asp114 and with the other amino acids closest to ligands in the binding cavity were calculated separately by density functional theory method in implicit water environment at the M06-2X/6-31 g(d,p) level of the theory. In a second approach, ligand binding affinities were calculated by taking into consideration not only the interaction energies but also deformation and desolvation energies of ligands with surrounding amino acid residues, in a radius of 5 Å of the protein-bound ligand. The quantum mechanically obtained results were compared with the experimentally obtained binding affinity values. We concluded that although H-bond interactions of ligands with Asp114 are the most dominant interaction in the binding site, if van der Waals and steric interactions of ligands which have cumulative effect on the ligand binding are not included in the calculations, the interaction energies are overestimated.     

Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.     

Insulin-Like Growth Factor (IGF) Binding Protein 2 Functions Coordinately with Receptor Protein Tyrosine Phosphatase β and the IGF-I Receptor To Regulate IGF-I-Stimulated Signaling

Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development and progression of atherosclerosis. IGF binding proteins (IGFBPs) modify IGF-I actions independently of IGF binding, but a receptor-based mechanism by which they function has not been elucidated. We investigated the role of IGFBP-2 and receptor protein tyrosine phosphatase β (RPTPβ) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPβ, which led to its dimerization and inactivation. This enhanced PTEN tyrosine phosphorylation and inhibited PTEN activity. Utilization of substrate trapping and phosphatase-dead mutants showed that RPTPβ bound specifically to PTEN and dephosphorylated it. IGFBP-2 knockdown led to decreased PTEN tyrosine phosphorylation and decreased AKT Ser473 activation. IGFBP-2 enhanced IGF-I-stimulated VSMC migration and proliferation. Analysis of aortas obtained from IGFBP-2^−/− mice showed that RPTPβ was activated, and this was associated with inhibition of IGF-I stimulated AKT Ser473 phosphorylation and VSMC proliferation. These changes were rescued following administration of IGFBP-2. These findings present a novel mechanism for coordinate regulation of IGFBP-2 and IGF-I signaling functions that lead to stimulation of VSMC proliferation. The results have important implications for understanding how IGFBPs modulate the cellular response to IGF-I.     

Characterization and Immunotherapeutic Implications for a Novel Antibody Targeting Interleukin (IL)-13 Receptor α2

The high affinity interleukin-13 receptor α2 (IL13Rα2) is selectively expressed at a high frequency by glioblastoma multiforme (GBM) as well as several other tumor types. One approach for targeting this tumor-specific receptor utilizes the cognate ligand, IL-13, conjugated to cytotoxic molecules. However, this approach lacks specificity because the lower affinity receptor for IL-13, IL13Rα1, is widely expressed by normal tissues. Here, we aimed to develop and characterize a novel monoclonal antibody (mAb) specific to IL13Rα2 for the therapeutic purpose of targeting IL13Rα2-expressing tumors. Hybridoma cell lines were generated and compared for binding affinities to recombinant human IL13Rα2 (rhIL13Rα2). Clone 47 demonstrated binding to the native conformation of IL13Rα2 and was therefore chosen for further studies. Clone 47 bound specifically and with high affinity (K(D) = 1.39 × 10(-9) m) to rhIL13Rα2 but not to rhIL13Rα1 or murine IL13Rα2. Furthermore, clone 47 specifically recognized wild-type IL13Rα2 expressed on the surface of CHO and HEK cells as well as several glioma cell lines. Competitive binding assays revealed that clone 47 also significantly inhibited the interaction between human soluble IL-13 and IL13Rα2 receptor. Moreover, we found that N-linked glycosylation of IL13Rα2 contributes in part to the interaction of the antibody to IL13Rα2. In vivo, the IL13Rα2 mAb improved the survival of nude mice intracranially implanted with a human U251 glioma xenograft. Collectively, these data warrant further investigation of this novel IL13Rα2 mAb with an emphasis on translational implications for therapeutic use.