Electrophiles produced during oxidative stress trigger pain responses by reacting with TRPA1 ion channels on sensory nerves. Bahia et al. show that residue C621 on TRPA1 has remarkable reactivity with electrophiles—more than cellular antioxidants—and is crucial for this sensory response.

PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. Abbreviations AKT V-Akt Murine Thymoma Viral Oncogene CA Cancer adjacent CDK1 Cyclin dependent kinase 1 CENPC-C Centromere Protein C ChIP Chromatin Immunoprecipitation co-IP Co-immunoprecipitation COSMIC Catalog of Somatic Mutations In Cancer CREB cAMP Responsive Element Binding Protein C-tail Carboxy terminal tail E2F1 E2F Transcription Factor 1 ECIS Electric Cell-substrate Impedance Sensing EGFR Epidermal Growth Factor Receptor GSI Gamma Secretase Inhibitor HDAC1 Histone Deacetylase 1 HP1 Heterochromatin protein 1 KAP1/TRIM28 KRAB-Associated Protein 1/Tripartite Motif Containing 28 MAF1 Repressor of RNA polymerase III transcription MAF1 homolog MCM2 Minichromosome Maintenance Complex Component 2 miRNA micro RNA MTF1 Metal-Regulatory Transcription Factor 1 PARP Poly(ADP-Ribose) Polymerase PD-1 Programmed Cell Death 1 PD-L1 Programmed Cell Death 1 Ligand 1 PI3K Phosphatidylinositol-4,5-Bisphosphate 3-Kinase PLK Polo-like Kinase pPTEN Phosphorylated PTEN PTEN Phosphatase and Tensin Homolog deleted on chromosome ten PTM Post Translational Modification Rad51 RAD51 Recombinase Rad52 RAD52 Recombinase RPA1 Replication protein A SILAC Stable Isotope Labeling with Amino Acids in Cell Culture SRF Serum Response Factor TKI Tyrosine Kinase inhbitors TMA Tissue Microarray TOP2A DNA Topoisomerase 2A.

Key pointsr We investigated the cardiovascular and respiratory responses of the normotensive Wistar-Kyoto (WKY) rat and the spontaneously hypertensive (SH) rat to inhalation and intravenous injection of the noxious stimuli allyl isothiocyanate (AITC).r AITC inhalation evoked atropine-sensitive bradycardia in conscious WKY rats, and evoked atropine-sensitive bradycardia and atenolol-sensitive tachycardia with premature ventricular contractions (PVCs) in conscious SH rats.r Intravenous injection of AITC evoked bradycardia but no tachycardia/PVCs in conscious SHs, while inhalation and injection of AITC caused similar bradypnoea in conscious SH and WKY rats.r Anaesthesia (inhaled isoflurane) inhibited the cardiac reflexes evoked by inhaled AITC but not injected AITC.r Data indicate the presence of a de novo nociceptive pulmonary-cardiac reflex triggering sympathoexcitation in SH rats, and this reflex is dependent on vagal afferents but is not due to steady state blood pressure or due to remodelling of vagal efferent function.Abstract Inhalation of noxious irritants/pollutants activates airway nociceptive afferents resulting in reflex bradycardia in healthy animals. Nevertheless, noxious pollutants evoke sympathoexcitation (tachycardia, hypertension) in cardiovascular disease patients. We hypothesize that cardiovascular disease alters nociceptive pulmonary-cardiac reflexes. Here, we studied reflex responses to irritants in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats. Inhaled allyl isothiocyanate (AITC) evoked atropine-sensitive bradycardia with atrial-ventricular (AV) block in conscious WKY rats, thus indicating a parasympathetic reflex. Conversely, inhaled AITC in conscious SH rats evoked complex brady-tachycardia J. Shane Hooper completed his BS in biology at the University of South Carolina and attaining a Professional Science Masters in biotechnology at the University of South Florida led him to realize that his scientific career would be in the field of research. He stayed at USF to complete his PhD studying arrhythmia evoked by airway nociceptive reflexes in healthy and cardiovascular diseased rats. He has learned that his true passion is in doing in vivo research and perfecting the required surgical procedures. His biggest challenge and future aspirations are to understand how cardiovascular disease alters neuronal interactions between the lungs and the heart. 3256 J. S. Hooper and others J Physiol 597.13with both AV block and premature ventricular contractions (PVCs). Atropine abolished the bradycardia and AV block, but the atropine-insensitive tachycardia and PVCs were abolished by the β 1 -adrenoceptor antagonist atenolol. The aberrant AITC-evoked reflex in SH rats was not reduced by acute blood pressure reduction by captopril. Surprisingly, intravenous AITC only evoked bradycardia in conscious SH and WKY rats. Furthermore, anaesthesia reduced the cardiac reflexes evoked by inhaled but not injected AITC. Nevertheless, anaesthesia had little effect on AITC-evoked respiratory ...

Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type ‘responding’ A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in ‘responding’ A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.

Inflammation causes activation of nociceptive sensory nerves, resulting in debilitating sensations and reflexes. Inflammation also induces mitochondrial dysfunction through multiple mechanisms. Sensory nerve terminals are densely packed with mitochondria, suggesting that mitochondrial signaling may play a role in inflammation-induced nociception. We have previously shown that agents that induce mitochondrial dysfunction, such as antimycin A, activate a subset of nociceptive vagal sensory nerves that express transient receptor potential (TRP) channels ankyrin 1 (A1) and vanilloid 1 (V1). However, the mechanisms underlying these responses are incompletely understood. Here, we studied the contribution of TRPA1, TRPV1 and reactive oxygen species (ROS) to antimycin A-induced vagal sensory nerve activation in dissociated neurons and at the sensory terminals of bronchopulmonary C-fibers. Nociceptive neurons were defined chemically and genetically. Antimycin A-evoked activation of vagal nociceptors in a Fura2 Ca 2+ assay correlated with TRPV1 responses compared to TRPA1 responses. Nociceptor activation was dependent on both TRP channels, with TRPV1 predominating in a majority of responding nociceptors and TRPA1 predominating only in nociceptors with the greatest responses. Surprisingly, both TRPA1 and TRPV1 were activated by H 2 O 2 when expressed in HEK293. Nevertheless, targeting ROS had no effect of antimycin A-evoked TRPV1 activation in either HEK293 or vagal neurons. In contrast, targeting ROS inhibited antimycin A-evoked TRPA1 activation in HEK293, vagal neurons and bronchopulmonary C-fibers, and a ROS-insensitive TRPA1 mutant was completely insensitive to antimycin A. We therefore conclude that mitochondrial dysfunction activates vagal nociceptors by ROS-dependent (TRPA1) and ROSindependent (TRPV1) mechanisms.