The dichotomous role of epiregulin in pain

Abstract

It has recently been shown that epidermal growth factor receptor (EGFR) contributes to the pathogenesis of pain. We scanned genetic markers within genes coding for receptors of the EGFR family (EGFR, ERBB2, ERBB3, and ERBB4) and their ligands (AREG, BTC, EGF, EPGN, EREG, HBEGF, MUC4, NRG1, NRG2, NRG3, NRG4, and TGFA) for association with self-reported pain intensity in patients with chronic facial pain who participated in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) cohort. We found that only epiregulin (EREG) was associated with pain. The strongest effect was observed for a minor allele at rs6836436 in EREG, which was associated with lower chronic pain intensity. However, the same allele was associated with higher facial pain intensity among cases with recent onset of facial pain. Similar trends were observed in an independent cohort of UK Biobank (UKB) where the minor allele at rs6836436 was associated with a higher number of acute pain sites but a lower number of chronic pain sites. Expression quantitative trait loci analyses established rs6836436 as a loss-of-function variant of EREG. Finally, we investigated the functional role of EREG using mouse models of chronic and acute pain. Injecting mice with an EREG monoclonal antibody reversed established mechanosensitivity in the complete Freund's adjuvant and spared nerve injury models of chronic pain. However, the EREG monoclonal antibody prolonged allodynia when administered during the development of complete Freund's adjuvant-induced mechanosensitivity and enhanced pain behavior in the capsaicin model of acute pain.

Figure 1:

Selection of acute and chronic pain phenotypes in the (A) OPPERA and (B) UKB cohorts.

Figure 2:

Quantile-quantile plot of 2,407 SNPs in genes coding for EGFR superfamily receptors and ligands, showing a significant association (FDR < 0.05) between seven EREG SNPs and chronic characteristic pain intensity (CPI) in OPPERA cohort.

Figure 3:

The EREG gene has two minor haplotypes. (A) Regional plot of EREG. (B) Illustration of the 16 SNPs in EREG Linkage Disequilibrium plot, numbers inside each cell indicate r² values, color reflects D’ value, ranging from white to red, (i.e. 0 to 1). (C) The sequence of three haplotypes with frequency > 5% within EREG gene locus. Major and minor alleles of EREG SNPs genotyped in OPPERA, SNPs significantly associated with CPI from Figure 2 are highlighted in green. Marker SNPs, namely, rs1993665, rs2367707, and rs6836436, for haplotypes H1, H2, and H3, respectively, are in highlighted in yellow.

Figure 4:

H3 and H2 haplotypes of EREG protects from chronic clinical pain. (A-B) OPPERA cohort. (A) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among chronic TMD cases and controls and (B) Plot of mean chronic pain intensity at baseline for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the OPPERA cohort. (C-D) UKB cohort. (C) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among chronic pain cases (at least one chronic pain site) and controls and (D) Plot of mean number of chronic pain sites for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the UKB cohort. Symbols represent mean ± SEM; False Discovery Rates (FDR) were derived by generalized linear modelling for haplotype association; *FDR < 0.05; **FDR < 0.01.

Figure 5:

H3 haplotype of EREG is a risk for acute clinical pain. (A-B) OPPERA cohort. (A) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among acute facial pain cases and controls and (B) Plot of mean of acute pain intensity at follow-up in controls for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the OPPERA cohort. (C-D) UKB cohort. (C) Bar plot of average minor allele counts of rs1993665, rs2367707, and rs6836436, (markers for haplotypes H1, H2, and H3, respectively) among acute pain cases (at least one acute pain site) and controls. (D) A plot of the mean number of acute pain sites for minor allele counts of rs1993665, rs2367707, and rs6836436, (markers for haplotypes H1, H2, and H3, respectively) in the UK biobank (UKB) cohort. Symbols represent mean ± SEM; False Discovery Rates (FDR) were derived by generalized linear modelling for haplotype association; *FDR < 0.05; **FDR < 0.01.

Figure 6:

The effects of systemically administering an EREG monoclonal antibody (mAb, 5 μg) in mouse models of pain. (A) Mice injected with the EREG mAb 3 days following CFA, as indicated by the arrow have higher paw withdrawal thresholds (g/mm²) compared with control mice 5 days post-CFA; n = 8/group. (B) The EREG mAb or vehicle control was administered 14 days following SNI surgery, as indicated by the arrow. A single administration of the mAb reverses mechanical allodynia for up to one week. (C) The concentration of EREG mAb in the blood plasma of mice following a single tail vein administration; n = 4/group. (D) Mice injected with the EREG mAb 1 day following CFA, as indicated by the arrow have lower paw withdrawal thresholds (g/mm²) than control mice 7 days post-CFA; n = 8/group. (E) Pre-treatment with the EREG mAb, 2 days before testing increases nocifensive behavior in the intraplantar capsaicin test of acute pain; n = 18–20/group. (F) A subset of mice from E were tested for mechanosensitivity following intraplantar capsaicin injection; n = 8/group. Mice injected with the EREG mAb have lower paw withdrawal thresholds (g/mm²) in the capsaicin injected paw, but not the uninjected paw when compared with controls. BL: baseline.*p < 0.05; **p < 0.001; ***p < 0.001 compared with vehicle at the indicated time points. †p < 0.05; ††p < 0.001 compared with capsaicin injected paw in F.