Spinal caspase-6 contributes to remifentanil-induced hyperalgesia via regulating CCL21/CXCR3 pathway in rats
Authors: Wang, Chunyan; Li, Qing; Jia, Zhen; Zhang, Haifang; Li, Yize; Zhao, Qi; Su, Lin; Yu, Yang; Xu, Rubin
Background: Neuroinflammation in the spinal cord is a pathological event in remifentanil-induced hyperalgesia (MD, but its underlying molecular mechanisms remain unclear. Recent studies recapitulate the significance of the intracellular protease caspase-6 in the release of inflammatory mediators and synaptic plasticity in pathologic pain. Also, chemokine CCL21 is involved in microglia activation and nociceptive transduction. This study examined whether spinal caspase-6 is associated with RIH via CCL21 and its receptor CXCR3. Methods: The acute exposure to remifentanil (1 mu g kg(-1)min(-1) for 60 min) was used to establish RIH, verified by assessment of mechanical paw withdrawal threshold and thermal paw withdrawal latency. The caspase-6 inhibitor, a neutralizing antibody against CCL21 (anti-CCL21), a selective CXCR3 antagonist NBI-74330, recombinant caspase-6 and CCL21 were used for the investigation of pathogenesis as well as the prevention of hyperalgesia. The expression of caspase-6, CCL21 and CXCR3 was also evaluated by RT-qPCR and Western blot. Results: This study discovered mechanical allodynia and thermal hyperalgesia along with the increase in the expression of spinal caspase-6 and CCL21/CXCR3 after remifentanil exposure. Central caspase-6 inhibition prevented behavioral RIH and spinal up-regulation of CCL21/CXCR3 level. Intrathecal anti-CCL21 injection reduced RIH and spinal expression of CXCR3. The delivery of recombinant caspase-6 facilitated acute nociceptive hypersensitivity and increased spinal CXCR3 release in naive rats, reversing by co-application of antiCCL21. Also, NBI-74330 attenuated RIH and exogenous CCL21-caused acute pain behaviors. Conclusion: This study highlighted that spinal caspase-6-mediated up-regulation of CCL21/CXCR3 is vital in the pathogenesis of RIH in rats.
Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, Which Is Compensated by the Release of Its Pro-chemotactic C-terminal Fragment
JOURNAL OF BIOLOGICAL CHEMISTRY
Authors: Shao, Zhifei; Morser, John; Leung, Lawrence L. K.
Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin-and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (RSKSKKFRR176)-R-168 sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.