Research ArticlePain

Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway

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Science Signaling  05 May 2020:
Vol. 13, Issue 630, eaaz1854
DOI: 10.1126/scisignal.aaz1854
  • Fig. 1 Spinal cord Hsp90 inhibition enhances morphine antinociception.

    (A to E) Male and female CD-1 mice were treated as indicated in the labels with either 17-AAG (0.5 nmol) or KU-32 [0.01 nmol (C)] or vehicle (Veh) injected by the intracerebroventricular (icv) or intrathecal (it) route, followed by a 24-hour recovery, then injected subcutaneously with or without morphine [Mor; 3.2 mg/kg (A to D); 3.2 or 10 mg/kg (E)], and subjected to behavioral testing. BL. baseline response. Data are means ± SEM from N (number of mice per group noted on each graph); each experiment was performed with one (D), two (A to C), or three (E) independent technical replicates (meaning groups of mice performed on different days). *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus same time point in Vehicle group, by two-way ANOVA with Sidak’s post hoc test.

  • Fig. 2 Brain Hsp90 inhibition overrides spinal cord Hsp90 inhibition with respect to opioid antinociception.

    (A and B) Representative images (A) and analysis (B) of Western blotting for Hsp70 in the periaqueductal gray (PAG) and spinal cord (SC) from male and female CD-1 mice that received intraperitoneal (ip) injection with 17-AAG (50 mg/kg) or vehicle with a 24-hour recovery. Hsp70 densitometry was normalized to that of GAPDH (loading control) from each sample and was further normalized to the vehicle group within each tissue. Data are means ± SEM of N = 9 to 10 mice, performed with two technical replicates. **P < 0.01 versus same tissue in vehicle group, by unpaired two-tailed t test. (C and D) Tail-flick (C) and paw incision (D) pain behavior tests in mice injected with 17-AAG (50 mg/kg) or vehicle intraperitoneally, followed by a 24-hour recovery and then a subcutaneous injection of morphine (3.2 mg/kg). Data are means ± SEM of N (number of mice per group), noted on each graph, performed with four (C) or two (D) technical replicates. *P < 0.05 and ****P < 0.0001 versus same time point in the 17-AAG group, by two-way ANOVA with Sidak’s post hoc test. (E and F) Tail-flick (E) and paw incision (F) pain behavior tests in mice that received both intracerebroventricular and intrathecal injections of 0.5 nmol of 17-AAG or vehicle, followed by a 24-hour recovery and then a subcutaneous injection of morphine (3.2 mg/kg). Data are means ± SEM of N (number of mice per group), noted on each graph, performed with two technical replicates. *P < 0.05 and ****P < 0.0001 versus same time point in the 17-AAG group, by two-way ANOVA with Sidak’s post hoc test.

  • Fig. 3 Spinal Hsp90 inhibition enables opioid activation of ERK MAPK signaling, leading to enhanced antinociception.

    (A to C) Representative images (A) and analysis (B and C) of Western blotting for phosphorylated ERK (pERK) and Hsp70 in the spinal cord from male and female CD-1 mice intrathecally injected with 0.5 nmol of 17-AAG or vehicle followed by a 24-hour recovery and then 0.1 nmol of DAMGO or vehicle intrathecally for 10 min. Densitometry of pERK was normalized to that of total ERK (tERK) within each sample, and the densitometry of Hsp70 was normalized to that of GAPDH; each was further normalized to that in the Vehicle/Vehicle group. Data are means ± SEM from N (the number of mice per group noted on the graphs), each performed as four technical replicates. In (B), *P < 0.05 and ****P < 0.0001 versus Vehicle/Vehicle group and ##P < 0.01 versus 17-AAG/Vehicle group (both by two-way ANOVA with Tukey’s post hoc test). In (C), P > 0.05 by unpaired two-tailed t test. (D) Immunohistochemistry (IHC) for pERK (green) performed on L4-L6 region spinal cord tissue from mice treated as described in (A). Representative images from N = 9 to 10 mice per group are shown. (E and F) Assessment of colocalization (yellow staining; white arrow) of pERK (green) with neuronal markers NeuN or MAP2 (red) by IHC of the dorsal horn region from 17-AAG/DAMGO-treated mice. Representative images from N ≥ 3 individual spinal cords per target, each performed as two independent technical replicates. Higher-magnification images (63×) are shown in (F). (G) Quantitation of the pERK signal in the dorsal horn region from all four groups in (D). Intensity values were normalized to the Vehicle/Vehicle group. N = 9 to 10 mice per group, each performed in four independent technical replicates. **P < 0.01 versus Vehicle/Vehicle group by two-way ANOVA with Tukey’s post hoc test. (H and I) Tail-flick (H) and paw incision (I) pain behavior tests in mice intrathecally treated with 0.5 nmol of 17-AAG or vehicle for 24 hours, followed by 5 μg of U0126 or vehicle intrathecally for 15 min, followed by morphine (3.2 mg/kg, subcutaneously). Data are means ± SEM from N (the number of mice per group noted on the graphs), each performed as four (H) or three (I) independent technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus same time point in the Veh/Veh group by two-way ANOVA with Sidak’s post hoc test.

  • Fig. 4 Spinal Hsp90 inhibition evokes rapid protein translation that mediates enhanced morphine-evoked antinociception.

    (A) Tail-flick assay on male and female CD-1 mice intrathecally injected with 0.5 nmol of 17-AAG or vehicle for 24 hours, then 85 nmol of cycloheximide (CX) or vehicle intrathecally for 30 min, and then morphine (3.2 mg/kg, subcutaneously). *P < 0.05, **P < 0.01, and ***P < 0.001 versus corresponding Veh/Veh data; #P < 0.05 and ##P < 0.01 versus corresponding 17-AAG/CX data (by two-way ANOVA with Sidak’s post hoc test). Data are means ± SEM from N (the number of mice per group as noted in the graph), each performed as four technical replicates. (B and C) Western blotting and densitometry analysis of pERK abundance in the spinal cords of mice intrathecally treated with 17-AAG then CX or vehicle as in (A), followed by 0.1 nmol of DAMGO or vehicle for 10 min. pERK density was normalized to tERK density in each sample and further normalized to the 17-AAG/Vehicle/Vehicle group. *P < 0.05 versus 17-AAG/Vehicle/Vehicle and #P < 0.05 versus 17-AAG/CX/Vehicle (both by two-way ANOVA with Tukey’s post hoc test). Data are means ± SEM from N (the number of mice per group as noted in the graph), each performed as six technical replicates.

  • Fig. 5 Quantitative proteomic analysis reveals a protein network altered by spinal Hsp90 inhibition.

    (A) Protein sample preparation and proteomic analysis workflow, as detailed in Materials and Methods. The samples were prepared using female CD-1 mice (N = 3 per group), which were intrathecally injected with 0.5 nmol of 17-AAG or vehicle for 24 hours. Spinal cords were removed for proteomic analysis, and protein was extracted as for Western blotting (detailed in Materials and Methods). These samples were used for all subsequent analysis in this figure. (B) Unbiased hierarchical clustering and heat map analysis of proteins significantly altered by 17-AAG treatment (P < 0.05). Red, increased; green, decreased; rows, individual proteins; columns, individual samples. Protein quantity traces for all proteins in each sample are shown (right insets). (C) Protein quantity data for the protein kinase RSK2, shown as means ± SEM of N = 3 per group. *P < 0.05 by unpaired two-tailed t test. (D) Principal components analysis of the full proteomic dataset was performed. Both treatment groups cluster together and are well separated along component 1, accounting for 75.1% of the variance. Within-group variance only occurs along component 2, accounting for only 8.5% of the variance. (E) Volcano plot of all detected proteins from the full proteomic dataset, plotting P value versus fold change. Red, significantly down-regulated; blue, significantly up-regulated; gray, not significant. (F) Gene ontology (GO) and KEGG pathway analysis of significantly altered proteins from (B) (see Materials and Methods for details). Data are plotted as significance versus fold enrichment. JNK, c-Jun N-terminal kinase.

  • Fig. 6 Spinal Hsp90 inhibition activates RSK1/2 phosphorylation, which mediates enhanced morphine-evoked antinociception.

    (A) Tail-flick assay in male and female CD-1 mice intrathecally injected with 0.5 nmol of 17-AAG or vehicle for 24 hours, followed by 10 nmol of Fmk or vehicle intrathecally for 30 min and then by morphine (3.2 mg/kg, subcutaneously). Data are means ± SEM from N (number of mice per group noted in the graph), each as three technical replicates. **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus same time point Vehicle/Vehicle group by two-way ANOVA with Sidak’s post hoc test. (B to D) Western blotting for phosphorylated (p) and total (t) RSK1 and RSK2 in the spinal cords from mice intrathecally injected with 0.5 nmol of 17-AAG or Vehicle for 24 hours, followed by 0.1 nmol of DAMGO or Vehicle intrathecally for 10 min. Densitometry of pRSK1 (C) and pRSK2 (D) was normalized to the corresponding tRSK within each sample and further normalized to the Vehicle/Vehicle group. Data are means ± SEM from N (number of mice per group noted in the graph), each as three technical replicates. *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus Vehicle/Vehicle group and ##P < 0.01 versus 17-AAG/Vehicle group (both by two-way ANOVA with Tukey’s post hoc test).

  • Fig. 7 Proposed model of Hsp90 regulation of opioid signaling in the spinal cord.

    Our data suggest that phosphorylation of ERK-MAPK proteins in the spinal cord by the MOR in response to opioids is blocked by Hsp90. Thus, Hsp90 inhibition (by 17-AAG or KU-32) enables ERK MAPK phosphorylation by the MOR with opioid treatment, leading to an ERK-RSK-translation cascade that promotes opioid antinociception.

Supplementary Materials

  • The PDF file includes:

    • Legends for data files S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.csv format). Full set of proteomic analysis results
    • Data file S2 (Microsoft Excel format). Individual proteomic analysis results for Gene Ontology/KEGG analysis.

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