DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

Spinal CCK1 Receptors Contribute to Somatic Pain Hypersensitivity Induced by Malocclusion via a Reciprocal Neuron-Glial Signaling Cascade.

Recent studies have shown that the incidence of chronic primary pain including temporomandibular disorders (TMD) and fibromyalgia syndrome (FMS) often exhibit comorbidities. We recently reported that central sensitization and descending facilitation system contributed to the development of somatic pain hypersensitivity induced by orofacial inflammation combined with stress. The purpose of this study was to explore whether TMD caused by unilateral anterior crossbite (UAC) can induce somatic pain hypersensitivity, and whether the cholecystokinin (CCK) receptor-mediated descending facilitation system promotes hypersensitivity through neuron-glia cell signaling cascade. UAC evoked thermal and mechanical pain hypersensitivity of the hind paws from day 5 to 70 that peaked at week 4 post UAC. The expression levels of CCK1 receptors, interleukin-18 (IL-18) and IL-18 receptors (IL-18R) were significantly up-regulated in the L4 to L5 spinal dorsal horn at 4 weeks post UAC. Intrathecal injection of CCK1 and IL-18 receptor antagonists blocked somatic pain hypersensitivity. IL-18 mainly co-localized with microglia, while IL-18R mainly co-localized with astrocytes and to a lesser extent with neurons. These findings indicate that the signaling transduction between neurons and glia at the spinal cord level contributes to the descending pain facilitation through CCK1 receptors during the development of the comorbidity of TMD and FMS. PERSPECTIVE: CCK1 receptor-dependent descending facilitation may mediate central mechanisms underlying the development of widespread somatic pain via a reciprocal neuron-glial signaling cascade, providing novel therapeutic targets for the clinical treatment of TMD and FMS comorbidities.

Spinal CCK contributes to somatic hyperalgesia induced by orofacial inflammation combined with stress in adult female rats.

In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK1 receptors, CCK2 receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK2 receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK1 receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK2 receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK2 receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK2 receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK2 receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.