Inhibition of heat shock protein 90 in the mouse spinal cord enhances opioid antinociception through upregulation of PKCβ in CGRP neurons.

Abstract

Decades of research have uncovered the complex signaling network downstream of the opioid receptors and suggested how this signaling could be modulated to improve opioid therapy. In our study, we have found that heat shock protein 90 (Hsp90) regulates downstream opioid signaling oppositely in the brain vs the spinal cord. In the spinal cord, we have found that Hsp90 inhibition enables antinociceptive signaling and disables pronociceptive signaling to enhance opioid pain relief and reduce side effects. We have now extended this study to analyze the contribution of protein kinase C (PKC) to the opioid signaling cascade. We used the Hsp90 inhibitor 17-AAG along with a PKC activator or inhibitor (Go6983) delivered into the spinal cords of male and female CD-1 mice to show that pan-PKC signaling contributes to the enhanced opioid antinociception observed in tail flick and postsurgical pain models. We used Western blot and immunohistochemistry to observe increased pan-PKC phosphorylation across calcitonin gene-related peptide (CGRP) and IB4 nociceptors in the spinal dorsal horn. We then used selective siRNA to identify PKCβ as the active isoform and further found PKCβ to be selectively activated in CGRP neurons by Hsp90 inhibition and morphine combined. Finally, we used cell-type-selective Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to knock down PKCβ in CGRP neurons and showed that this specific isoform in these specific cells was necessary for enhanced opioid antinociception after Hsp90 inhibition.Together, these studies further uncover the novel Hsp90-regulated opioid signaling cascade and suggest how Hsp90 inhibitors could be used to improve opioid therapy by increasing analgesic efficacy and decreasing side effects.