The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

The proteasome inhibitor bortezomib (BTZ) is a potent first-line anticancer drug for multiple myeloma; nonetheless, it induced peripheral neuropathy. It has been suggested that many cytokines may play a role in mediating neuropathic pain, but the underlying molecular mechanism is not fully understood. Recent studies have shown that neuropathic pain is closely related to the purinergic ligand-gated ion channel 7 receptor (P2X7R), one of the P2X receptors, which is richly expressed in glial cells. P2X7-p38 pathway is correlated with microglia- and satellite glial cell- (SGC-) mediated neuropathic pain. However, the association of P2X7R and p38MAPK in mediating BTZ-induced neuropathic pain remains unclear. In this study, the relationship between P2X7R activation and p38 phosphorylation in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) in the development and maintenance of BTZ-induced neuropathic pain was elucidated. The results showed that BTZ increased mechanical thresholds in rats, accompanied with upregulation of P2X7R expression and p38MAPK phosphorylation, indicating that P2X7R and p38MAPK are key molecules in the development and maintenance of BTZ-induced neuropathic pain. Inhibiting p38MAPK phosphorylation with SB203580 resulted in downregulation of P2X7R expression levels. Inhibition of P2X7R with Brilliant Blue G (BBG) reversed neuropathic pain might decrease through the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 via inhibiting p38MAPK phosphorylation. The P2X7R/p38MAPK signaling pathway in SGCs of DRG and microglia of SDH might be a potential pharmacological target behind this mechanism as an opportunity to relieve BTZ-induced neuropathic pain.

Deletion of P2X7 Receptor Decreases Basal Glutathione Level by Changing Glutamate-Glutamine Cycle and Neutral Amino Acid Transporters

Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate–glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine–cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

The role of the P2X7 nucleotide receptor in salivary gland inflammation

Salivary gland inflammation is a hallmark of Sjogren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration and the consequent impairment of the salivary gland and loss of saliva secretion, predominantly in women. The current therapeutic management of SS is relatively ineffective and does not address the underlying inflammatory processes contributing to the pathology of SS. In this study, two novel therapeutic approaches were evaluated to limit salivary gland inflammation and improve secretory function, i.e., antagonism of the P2X7 nucleotide receptor (P2X7R), which prevents salivary gland inflammation and activation of the P2Y[subscript2] nucleotide receptor (P2Y[subscript2]R) which stimulates the regeneration of damaged salivary glands. The P2X7R is an ATP-gated non-selective cation channel that regulates inflammatory responses in cells and tissues, including salivary gland epithelium. The P2X7R contributes to the pathology of a variety of inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In immune cells, P2X7R activation induces the production of pro-inflammatory cytokines, including IL-1[beta] and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. This study (Chapter II) sheds light on the role of the P2X7R in salivary gland inflammation and hyposalivation. Our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1[beta], responses that are absent in SMG cells isolated from mice devoid of P2X7Rs (P2X7R[superscript-/-]). P2X7R-mediated IL-1[beta] release in SMG epithelial cells is dependent on downhill transmembrane Na[superscript+] and/or K[superscript+] fluxes, the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome, and the generation of mitochondrial ROS. In vivo administration of the P2X7R antagonist A438079 in the CD28[superscript-/-], IFN[superscript][superscript-/-], NOD.H-2[superscripth4] mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function. The P2Y[subscript2]R, a G protein-coupled receptor equipotently activated by ATP and UTP, is upregulated in a variety of tissues, including salivary gland epithelium, in response to injury or stress and is proposed to play a role in tissue regeneration. The results indicated that P2Y2R activation with UTP enhances the migration, aggregation and self-organization of dispersed salivary epithelial cells forming spheres that display characteristics similar to differentiated acini in salivary glands. One of the consequences of the chronic inflammatory disease SS is the fibrosis of the salivary gland. The role of transforming growth factor- [beta] (TGF-[beta]) is well established in the fibrosis and regeneration of various organs, including the liver, lung and kidney. In this study, results with a submandibular gland (SMG) duct ligation-induced mouse model of fibrosis indicated that 7 days of SMG duct ligation resulted in upregulation of TGF-β signaling components which correlated with the upregulation of the fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-[beta] receptor 1 inhibitors. These results suggest that TGF-[beta] signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis.

P2X7 receptors: role in bone cell formation and function

The role of the P2X7 receptor (P2X7R) is being explored with intensive interest in the context of normal bone physiology, bone-related diseases and, to an extent, bone cancer. In this review, we cover the current understanding of P2X7R regulation of bone cell formation, function and survival. We will discuss how the P2X7R drives lineage commitment of undifferentiated bone cell progenitors, the vital role of P2X7R activation in bone mineralisation and its relatively unexplored role in osteocyte function. We also review how P2X7R activation is imperative for osteoclast formation and its role in bone resorption via orchestrating osteoclast apoptosis. Variations in the gene for the P2X7R (P2RX7) have implications for P2X7R-mediated processes and we review the relevance of these genetic variations in bone physiology. Finally, we highlight how targeting P2X7R may have therapeutic potential in bone disease and cancer.

Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.