Epiregulin and EGFR interactions are involved in pain processing
Loren J. Martin, Shad B. Smith, Arkady Khoutorsky, Claire A. Magnussen, Alexander Samoshkin, Robert E. Sorge, Chulmin Cho, Noosha Yosefpour, Sivaani Sivaselvachandran, Sarasa Tohyama, Tiffany Cole, Thang M. Khuong, Ellen Mir, Dustin G. Gibson, Jeffrey S. Wieskopf, Susana G. Sotocinal, Jean Sebastien Austin, Carolina B. Meloto, Joseph H. Gitt, Christos Gkogkas, Nahum Sonenberg, Joel D. Greenspan, Roger B. Fillingim, Richard Ohrbach, Gary D. Slade, Charles Knott, Ronald Dubner, Andrea G. Nackley, Alfredo Ribeiro-da-Silva, G. Gregory Neely, William Maixner, Dmitri V. Zaykin, Jeffrey S. Mogil, Luda Diatchenko
Loren J. Martin, Shad B. Smith, Arkady Khoutorsky, Claire A. Magnussen, Alexander Samoshkin, Robert E. Sorge, Chulmin Cho, Noosha Yosefpour, Sivaani Sivaselvachandran, Sarasa Tohyama, Tiffany Cole, Thang M. Khuong, Ellen Mir, Dustin G. Gibson, Jeffrey S. Wieskopf, Susana G. Sotocinal, Jean Sebastien Austin, Carolina B. Meloto, Joseph H. Gitt, Christos Gkogkas, Nahum Sonenberg, Joel D. Greenspan, Roger B. Fillingim, Richard Ohrbach, Gary D. Slade, Charles Knott, Ronald Dubner, Andrea G. Nackley, Alfredo Ribeiro-da-Silva, G. Gregory Neely, William Maixner, Dmitri V. Zaykin, Jeffrey S. Mogil, Luda Diatchenko
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Research Article Neuroscience

Epiregulin and EGFR interactions are involved in pain processing

Abstract

The EGFR belongs to the well-studied ErbB family of receptor tyrosine kinases. EGFR is activated by numerous endogenous ligands that promote cellular growth, proliferation, and tissue regeneration. In the present study, we have demonstrated a role for EGFR and its natural ligand, epiregulin (EREG), in pain processing. We show that inhibition of EGFR with clinically available compounds strongly reduced nocifensive behavior in mouse models of inflammatory and chronic pain. EREG-mediated activation of EGFR enhanced nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. Moreover, EREG application potentiated capsaicin-induced calcium influx in a subset of sensory neurons. Both the EGFR and EREG genes displayed a genetic association with the development of chronic pain in several clinical cohorts of temporomandibular disorder. Thus, EGFR and EREG may be suitable therapeutic targets for persistent pain conditions.

Authors

Loren J. Martin, Shad B. Smith, Arkady Khoutorsky, Claire A. Magnussen, Alexander Samoshkin, Robert E. Sorge, Chulmin Cho, Noosha Yosefpour, Sivaani Sivaselvachandran, Sarasa Tohyama, Tiffany Cole, Thang M. Khuong, Ellen Mir, Dustin G. Gibson, Jeffrey S. Wieskopf, Susana G. Sotocinal, Jean Sebastien Austin, Carolina B. Meloto, Joseph H. Gitt, Christos Gkogkas, Nahum Sonenberg, Joel D. Greenspan, Roger B. Fillingim, Richard Ohrbach, Gary D. Slade, Charles Knott, Ronald Dubner, Andrea G. Nackley, Alfredo Ribeiro-da-Silva, G. Gregory Neely, William Maixner, Dmitri V. Zaykin, Jeffrey S. Mogil, Luda Diatchenko

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Figure 4

EREG and EGFR are upregulated in chronic pain states, and EREG increases activation of medium-small DRG sensory neurons.

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EREG and EGFR are upregulated in chronic pain states, and EREG increases...
(A) EREG in the blood is upregulated by CFA and SNI, but not formalin (F3,38 = 10.0, P < 0.001), as measured by ELISA. Bars represent mean ± SEM for protein levels (pg/ml); n = 9–10 biological replicates/group. One-way ANOVA followed by Dunnett’s case-comparison post hoc test. *P < 0.05; ***P < 0.001 compared with control group. (B) EGFR (green) is abundantly found in all DRG sensory neurons. Scale bar: 50 μm. (C) The cellular distribution of EGFR is equal among different cell sizes that exhibit either high or low EGFR staining. n = 3 mice. (D) Top: representative Western blots showing p-EGFR and β-actin in the DRG before (BL) (left band) and 3 days or 7 days after CFA or SNI, respectively (right band). Bottom: quantification of Western blot data (n = 5 biological replicates/condition) after normalization to β-actin and compared with baseline values. *P < 0.05 compared with 1.0 by 1-tailed t test (CFA: t4 = 2.5, P = 0.03; SNI: t4 = 3.3, P = 0.02). (E) Representative calcium traces of neurons responsive to multiple capsaicin (500 nM, 15 seconds for every 4 minutes) pulses and treated either with vehicle (HBSS, left panel) or EREG (200 ng/ml, right panel) for 6 minutes before 3 challenging pulses of capsaicin were applied. The ratio of Ca2+ peak heights (b/a) before and after exposure to EREG or vehicle was calculated as a measure of signal enhancement. (F) Collected b/a ratio values obtained from experiments in E. White bars show ratios obtained without exposure to EREG (ncell = 60, nexp = 9). The distribution was well fitted by a Gaussian function with mean of 0.76, SD of 0.12, and upper 95% 2-tailed confidence limit of 1.05 (arrow). Green bars show ratios following 6 minutes exposure to EREG (200 ng/ml; ncell = 101, nexp = 13). Following EREG exposure, 40.79% of ratios exceeded the 95% confidence limit, and the mean ± SEM of these ratio values was 1.25 ± 0.03. (G) Neurons treated with EREG were separated into 2 groups: those in which sensitization was (ncell = 40) or was not (ncell = 61) observed in the first exposure following EREG addition. Data presented as mean ± SEM, n = 40–61/group, from a total of 9/13 experiments for vehicle/EREG. ***P < 0.001, unpaired Student’s t test. (H) The proportion of neurons sensitized in the presence of EREG based on b/a ratio values greater than 2 SD above the mean of the vehicle group.