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(PS12) Oncostatin M induces hyperalgesic priming and amplifies signaling of cAMP to ERK by RapGEF2 and PKA


Andrea Ebersberger

Hans-Georg Schaible

Anibal Garza Carbajal

Jon Levine – University of California, San Francisco

Alina Thiel

Tim Hucho

Maike Siobal

Katharina Möller

Stephanie Brosig

Luiz Ferrari

Jeremy Acuna


Background and Aims : Hyperalgesic priming enhances and prolongs pain sensitization by agents such as prostaglandin E2 (PGE2). Mechanisms underlying priming have gained increased interest as one possible mechanism underlying chronification of pain. We set out to identify novel priming inducing stimuli and to analyze underlying cellular signaling mechanisms.

Methods : Signaling activity was monitored by a single-cell based high content screening microscopy approach. Rat DRG neurons were dissociated and cultured overnight in Neurobasal A medium in 96 well plates. After stimulation, cells were PFA fixed, blocked and immunocytochemically tested for signaling and/or subgroup markers. After digitalization with a Cellomics XTi automated microscope, software based automatic object identification allowed unbiased single-cell quantification. Cellular aspects of priming were investigated by electrophysiological single-cell recordings. Priming in the animal was confirmed by intradermal injection of OSM into adult rat paws and subsequent Randall Selitto measurements of mechanical pain thresholds.

Results : We present the cytokine Oncostatin M (OSM), a member of the interleukin-6 family, to induce priming in adult male rats. Using a single-cell based high content screening microscopy approach on cultured sensory neurons (1-7), we found overnight incubation of primary sensory neurons with OSM to result in increased extracellular signal-reguated kinase (ERK) activation by agents inducing cyclic AMP (cAMP) production such as PGE2, forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons and required translational activity. OSM pretreatment rendered baseline currents ERK-dependent and abolished further current increase by ERK-independent mechanisms. Contrary to other priming models, OSM increased cAMP-to-ERK signaling without changing the pathway structure. In both, control and OSM-treated neurons, we found cAMP/ERK signaling to involve RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling was induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts rather on IB4/CaMKIIα-negative neurons.

Conclusions : Beyond introducing OSM as a novel priming-inducing substance, our results propose a novel mechanism for priming. Independent of so far described changes such as G-protein switch (8) or increased EPAC signaling (9-11), we find an increased output of pathways normally activating ERK downstream to cAMP production.

References : 1. Andres C, Meyer S, Dina OA, Levine JD, Hucho T. 2010. Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization. Mol Pain 6: 98
2. Isensee J, Diskar M, Waldherr S, Buschow R, Hasenauer J, Prinz A, Allgower F, Herberg FW, Hucho T. 2014. Pain modulators regulate the dynamics of PKA-RIIPKA-PRII phosphorylation in subgroups of sensory neurons. J Cell Sci 127: 216-29
3. Isensee J, Hucho T. 2017. HCS-Mikroskopie – ein Schlüssel zu intrazellulären Schmerzmechanismen. BIOspektrum 23: 277-80
4. Isensee J, Kaufholz M, Knape MJ, Hasenauer J, Hammerich H, Gonczarowska-Jorge H, Zahedi RP, Schwede F, Herberg FW, Hucho T. 2018. PKA-RIIPKA-PRII subunit phosphorylation precedes activation by cAMP and regulates activity termination. The Journal of Cell Biology
5. Isensee J, Schild C, Schwede F, Hucho T. 2017. Crosstalk from cAMP to ERK1/2 emerges during postnatal maturation of nociceptive neurons and is maintained during aging. J Cell Sci 130: 2134-46
6. Isensee J, Wenzel C, Buschow R, Weissmann R, Kuss AW, Hucho T. 2014. Subgroup-elimination transcriptomics identifies signaling proteins that define subclasses of TRPV1-positive neurons and a novel paracrine circuit. PLoS One 9: e115731
7. Isensee J, Krahe L, Moeller K, Pereira V, Sexton JE, Sun X, Emery E, Wood JN, Hucho T. 2017. Synergistic regulation of serotonin and opioid signaling contributes to pain insensitivity in Nav1.7 knockout mice. Sci Signal 10
8. Dina OA, Khasar SG, Gear RW, Levine JD. 2009. Activation of Gi induces mechanical hyperalgesia poststress or inflammation. Neuroscience 160: 501-7
9. Hucho TB, Dina OA, Levine JD. 2005. Epac mediates a cAMP-to-PKC signaling in inflammatory pain: an isolectin B4(+) neuron-specific mechanism. J Neurosci 25: 6119-26
10. Vasko MR, Habashy Malty R, Guo C, Duarte DB, Zhang Y, Nicol GD. 2014. Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac. PLoS One 9: e104529
11. Eijkelkamp N, Wang H, Garza-Carbajal A, Willemen HL, Zwartkruis FJ, Wood JN, Dantzer R, Kelley KW, Heijnen CJ, Kavelaars A. 2010. Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK. J Neurosci 30: 12806-15

Conflicts of Interest : None

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