Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy.

Homologous recombination (HR) serves multiple roles in DNA repair that are essential for maintaining genomic stability, including double-strand DNA break (DSB) repair. The central HR protein, RAD51, is frequently overexpressed in human malignancies, thereby elevating HR proficiency and promoting resistance to DNA-damaging therapies. Here, we find that the non-canonical NF-κB factors p100/52, but not RelB, control the expression of RAD51 in various human cancer subtypes. While p100/p52 depletion inhibits HR function in human tumor cells, it does not significantly influence the proficiency of non-homologous end joining, the other key mechanism of DSB repair. Clonogenic survival assays were performed using a pair DLD-1 cell lines that differ only in their expression of the key HR protein BRCA2. Targeted silencing of p100/p52 sensitizes the HR-competent cells to camptothecin, while sensitization is absent in HR-deficient control cells. These results suggest that p100/p52-dependent signaling specifically controls HR activity in cancer cells. Since non-canonical NF-κB signaling is known to be activated after various forms of genomic crisis, compensatory HR upregulation may represent a natural consequence of DNA damage. We propose that p100/p52-dependent signaling represents a promising oncologic target in combination with DNA-damaging treatments.

Extracellular Nucleotides and Histamine Suppress TLR3- and RIG-I-Mediated Release of Antiviral IFNs from Human Airway Epithelial Cells.

Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y2 and H1 receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.

Differential Regulation of ATP- and UTP-Evoked Prostaglandin E2 and IL-6 Production from Human Airway Epithelial Cells.

The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.

VCAM-1 induces signals that stimulate ZO-1 serine phosphorylation and reduces ZO-1 localization at lung endothelial cell junctions.

Endothelial cell VCAM-1 regulates recruitment of lymphocytes, eosinophils, mast cells, or dendritic cells during allergic inflammation. In this report, we demonstrated that, during allergic lung responses, there was reduced zonula occludens (ZO)-1 localization in lung endothelial cell junctions, whereas there was increased lung endothelial cell expression of VCAM-1, N-cadherin, and angiomotin. In vitro, leukocyte binding to VCAM-1 reduced ZO-1 in endothelial cell junctions. Using primary human endothelial cells and mouse endothelial cell lines, Ab crosslinking of VCAM-1 increased serine phosphorylation of ZO-1 and induced dissociation of ZO-1 from endothelial cell junctions, demonstrating that VCAM-1 regulates ZO-1. Moreover, VCAM-1 induction of ZO-1 phosphorylation and loss of ZO-1 localization at cell junctions was blocked by inhibition of VCAM-1 intracellular signals that regulate leukocyte transendothelial migration, including NOX2, PKCα, and PTP1B. Furthermore, exogenous addition of the VCAM-1 signaling intermediate H2 O2 (1 µM) stimulated PKCα-dependent and PTP1B-dependent serine phosphorylation of ZO-1 and loss of ZO-1 from junctions. Overexpression of ZO-1 blocked leukocyte transendothelial migration. In summary, leukocyte binding to VCAM-1 induces signals that stimulated ZO-1 serine phosphorylation and reduced ZO-1 localization at endothelial cell junctions during leukocyte transendothelial migration.

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics.

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.

Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules.

Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)-a key regulator of cardiovascular, urinary and CNS function-binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.