Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A) ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca(2+) imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4E(S209A) mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4E(S209A) and Mnk1/2(-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.
Environmental exposure to arsenic results in multiple adverse effects in the lung. Our objective was to identify potential pulmonary protein biomarkers in the lung-lining fluid of mice chronically exposed to low-dose As and to validate these protein changes in human populations exposed to As.
Protease-activated receptor-2 (PAR2) belongs to a four-member family of G-Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased-ligand signaling. We recently demonstrated that lipid tethering of known PAR2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency > 200 fold. In this study, we used lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2-aminothiazoyl; 2-at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR2 antagonists. Compound 562 (C562), 2-aminothiazol-4yl-SKGRS-PEG3-Hdc blocks PAR2 Ca2+ signaling elicited via peptidomimetics (2-at-LIGRL-NH2) or via asthma-associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o-). This compound was a biased-signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR2. A shortened version of C562, 2-at-SKGR-PEG3-Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR2 agonist, 2-at-LIGR-PEG3-Hdc. Thus, we screened a series of potential PAR2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG3-Hdc lipid tether. We describe several potent agonists, and one partial agonist (C608, 2-at-TIGR-PEG3-Hdc) that also acts as a potent, specific and biased signaling antagonist of PAR2. When used in nanomolar concentrations, C608 blocked PAR2-dependent Ca2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR2 full and biased ligand signaling.
Nanomaterials are increasingly used in a variety of industrial processes and consumer products. There are growing concerns about the potential impacts for public health and environment of engineered nanoparticles. The aim of this work was to evaluate a novel impedance-based real time cell analyzer (RTCA) as a high-throughput method for screening the cytotoxicity of nanoparticles and to validate the RTCA results using a conventional cytotoxicity test (MTT). A collection of 11 inorganic nanomaterials (Ag(0), Al(2)O(3), CeO(2), Fe(0), Fe(2)O(3), HfO(2), Mn(2)O(3), SiO(2), TiO(2), ZnO, and ZrO(2)) were tested for potential cytotoxicity to a human bronchial epithelial cell, 16HBE14o-. The data collected by the RTCA system was compared to results obtained using a more traditional methyl tetrazolium (MTT) cytotoxicity assay at selected time points following application of nanomaterials. The most toxic nanoparticles were ZnO, Mn(2)O(3) and Ag(0), with 50% response at concentrations lower than 75 mg/L. There was a good correlation in cytotoxicity measurements between the two methods; however, the RTCA method maintained a distinct advantage in continually following cytotoxicity over time. The results demonstrate the potential and validity of the impedance-based RTCA technique to rapidly screen for nanoparticle toxicity.