Select steroid hormone glucuronide metabolites can cause toll-like receptor 4 activation and enhanced pain

Abstract

We have recently shown that several classes of glucuronide metabolites, including the morphine metabolite morphine-3-glucuronide and the ethanol metabolite ethyl glucuronide, cause toll like receptor 4 (TLR4)-dependent signaling in vitro and enhanced pain in vivo. Steroid hormones, including estrogens and corticosterone, are also metabolized through glucuronidation. Here we demonstrate that in silico docking predicts that corticosterone, corticosterone-21-glucuronide, estradiol, estradiol-3-glucuronide and estradiol-17-glucuronide all dock with the MD-2 component of the TLR4 receptor complex. In addition to each docking with MD-2, the docking of each was altered by pre-docking with (+)-naloxone, a TLR4 signaling inhibitor. As agonist versus antagonist activity cannot be determined from these in silico interactions, an in vitro study was undertaken to clarify which of these compounds can act in an agonist fashion. Studies using a cell line transfected with TLR4, necessary co-signaling molecules, and a reporter gene revealed that only estradiol-3-glucuronide and estradiol-17-glucuronide increased reporter gene product, indicative of TLR4 agonism. Finally, in in vivo studies, each of the 5 drugs was injected intrathecally at equimolar doses. In keeping with the in vitro results, only estradiol-3-glucuronide and estradiol-17-glucuronide caused enhanced pain. For both compounds, pain enhancement was blocked by the TLR4 antagonist lipopolysaccharide from Rhodobacter sphaeroides, evidence for the involvement in TLR4 in the resultant pain enhancement. These findings have implications for several chronic pain conditions, including migraine and temporomandibular joint disorder, in which pain episodes are more likely in cycling females when estradiol is decreasing and estradiol metabolites are at their highest.

Keywords: (+)-Naloxone; Corticosterone; Corticosterone-21-glucuronide; Estradiol; Estradiol-17-glucuronide; Estradiol-3-glucuronide; TLR4.

Figure 1

Corticosterone caused a significant decrease in HEK-TLR4 cell SEAP reporter expression (F_(5,25)=9.05, p<0.05, A). Bonferroni post-hoc tests showed a significant decreased between vehicle and 10 (t=1.15, p<0.05), 1 (t=1.84, p<0.05), and 0.1 (t=1.84, p<0.05) μM corticosterone concentrations. CortG did not cause any changes in HEK-TLR4 SEAP expression compared to vehicle (F_(5,23)=1.72, p>0.05, B).

Figure 2

Estradiol caused a significant decrease in HEK-TLR4 cell SEAP reporter gene expression relative to 1% DMSO vehicle (F_{(5, 30)}=9.60, p<0.05, A). Bonferroni post-hoc tests showed a significant decrease from vehicle at 10 (t=3.696, p<0.05), 1 (t=4.14, p<0.05), 0.1 (t=3.61, p<0.05) and 0.01 (t=3.426, p<0.05) μM estradiol concentrations. E₂-3-G caused a significant increase in HEK-TLR4 cell NFκB-dependent SEAP expression relative to water (F=19.2, p<0.05, B). A Bonferroni post-hoc test showed E₂-3-G significantly increased SEAP expression at 100 (t=42.35, p<0.05), 10 (t=4.84, p<0.05), 1 (t=4.95, p<0.05), 0.1 (t=4.82, p<0.05) and 0.01 (t=3.505, p<0.05) μM E₂-3-G concentrations. E₂-17-G also caused a significant increase in HEK-TLR4 cell SEAP expression relative to 0.1% DMSO vehicle at the 100 μM concentration (F_(5,18)=72.81, p<0.05, Bonferroni post-hoc t=14.63, p<0.05, C).

Figure 3

The TLR4 inhibitor (+)-naloxone significantly attenuated the HEK-TLR4 cell SEAP increases caused by 100 μM E₂-3-G (F_(5,17)=6.89, p<0.05, A), or 100 μM E₂-17-G (F_(5,14)= 9.18, p<0.05, C) at the 1 and 10 μM concentrations of (+)-naloxone. Bonferroni post-hoc tests showed a significant decrease in SEAP expression when 100 μM E₂-3-G was incubated with 10 (t=4.49, p<0.05) or 1 μM (t=4.96, p<0.05) (+)-naloxone compared to 100 μM E₂-3-G alone. Similarly, Bonferroni post-hoc tests showed a significant decrease in SEAP expression when 100 μM E₂-17-G was incubated with 10 (t=5.06, p<0.05) or 1 μM (t=5.25, p<0.05) (+)-naloxone compared to 100 μM E₂-3-G alone. The TLR4 antagonist LPS-RS significantly attenuated the HEK-TLR4 cell SEAP increases caused by 100 μM E₂-3-G (F_(5,19)=5.22, p<0.05, B) or 100 μM E₂-17-G (F_(5,16)=7.81, p<0.05, D) at all three concentrations tested (0.1, 1 and 10 ng/ml). Bonferroni post-hoc tests showed a significant decrease in SEAP expression when 100 μM E₂-3-G was coincubated with 10 (t=4.29, p<0.05), 1 (t=4.38, p<0.05), or 0.1 (t=3.62, p<0.05) ng/ml LPS-RS. Similarly, Bonferroni post-hoc tests showed a significant decrease in SEAP expression when 100 μM E₂-17-G was coincubated with 10 (t=4.29, p<0.05), 1 (t=4.38, p<0.05), or 0.1 (t=3.62, p<0.05) ng/ml LPS-RS.

Figure 4

Intrathecal injection of corticosterone (0.56 μg, F_(2,22)=3.59, p<0.05, A), CortG (0.85 μg, F_(2,22)=0.93, p>0.05, B) or E₂ (0.44 μg, F_(2,22)=2.82, p>0.05, C) failed to cause significant allodynia relative to vehicle controls. Intrathecal injection of E₂-3-G (0.76 μg, F_{(4, 40)}=11.98, p<0.05, D) and E₂-17-G (0.76 μg, F_(4,34), p<0.05, E) caused significant allodynia to develop, which was blocked by LPS-RS (40 μg intrathecal) coadministration. Bonferroni post-hoc tests showed a significant increase in sensitivity was present at 1 (t=4.61, p<0.05) and 3 (t=8.04, p<0.05) hours following E₂-3-G administration. The allodynia caused by E₂-3-G injection was blocked at 1 (t=4.10, p<0.05) and 3 (t=7.89, p<0.05) hours by coadministration of 40 μg LPS- RS. Similarly, Bonferroni post-hoc tests showed a significant increase in sensitivity following E₂-17-G administration at 3 hours (t=3.99, p<0.05) which was blocked by LPS-RS (40 μg intrathecal; t=5.02, p<0.05) coadministration.