The Role of the P2X7 Receptor in Radiation-Induced Salivary Gland Inflammation and Dysfunction

Abstract

Annually, >600,000 cases of head and neck cancer are diagnosed worldwide, with treatment commonly entailing surgical removal of the tumor followed by ionizing radiation (IR) therapy. Salivary glands, located proximal to tumors, are incidentally damaged during radiotherapy, leading to loss of glandular function. Reduced saliva output leads to many health complications such as increased risk of mucositis, dental caries, and malnutrition, and makes talking, chewing, and swallowing difficult, greatly reducing the quality of life of patients. A limited number of therapeutic options for salivary hypofunction are currently available, however, they are palliative and require life-long use by patients. Therefore, uncovering the signaling mechanisms behind radiation-induced damage to salivary glands is critical to develop novel drug targets to reverse salivary gland dysfunction and, thus, improve the quality of life for head and neck cancer survivors. The P2X7 receptor (P2X7R) is a purinergic receptor activated by extracellular adenosine triphosphate (eATP, >100 μM), a damage-associated molecular pattern (DAMP) typically released from cells to alarm neighbors of a nearby insult. Brief P2X7R activation by eATP leads to formation of a homotrimeric, nonselective cation channel, whereas repetitive or prolonged stimulation causes formation of a large pore and activation of the NLRP3 inflammasome. P2X7R activation often leads to reactive oxygen species (ROS) generation, membrane depolarization, membrane blebbing, cytokine production, or cell death. To evaluate the possible role of the P2X7R in radiation-induced damage to salivary glands, mice that are globally deficient in the P2X7R (P2X7R-/-) were used for experiments and outputs were compared to wildtype (C57BL/6J) mice. Immediately following radiation exposure, there is increased ATP secreted from wildtype primary parotid glands cells that is not observed in P2X7R-/- cells. Surprisingly, this did not lead to alterations in cell death at 24-72 hours post-IR between genotypes. Irradiated wildtype primary parotid cells secrete high levels of the biologically active lipid, prostaglandin E2 (PGE2) that is not detected in P2X7R-/- cells and does not clearly align with mRNA levels and activity of PGE2 synthesizing enzymes. Remarkably, P2X7R-/- mice have improved salivary gland function at days 3 and 30 post-IR and treatment with a P2X7R-selective antagonist, A438079, was able to preserve salivary gland function in wildtype mice. This study suggests P2X7R antagonism may be a promising approach for preventing radiation-induced salivary gland dysfunction and further understanding of PGE2-mediated signaling in irradiated salivary glands is warranted. PGE2 is a bioactive lipid derived from arachidonic acid that signals via four E-prostanoid receptors (EP1-4R) to induce various physiological outputs. EP2 and EP4 canonically activate adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP) levels, leading to cAMP response element binding protein (CREB) phosphorylation. EP2 and EP4 also activate a non-canonical pathway of EGFR transactivation via β-arrestin that leads to protein kinase B/Akt phosphorylation. EP3 inhibits adenylate cyclase, decreasing cAMP-mediated signaling and CREB phosphorylation. EP1 activates phospholipase C, leading to increased inositol 1,4,5-triphosphate and calcium within the cell. EPRs have also been shown to activate other signaling pathways, including p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways, although the corresponding EPR is not clearly defined for these pathways. As described above, there is increased PGE2 in supernatants from irradiated wildtype primary parotid gland cells, with a significant reduction of PGE2 secreted from P2X7R-/- cells that correlates with improved salivary gland function in mice post-IR. Differences in PGE2-mediated pathways between wildtype and P2X7R-/- mice have not been previously described in irradiated parotid glands and is the major goal of this study. EPR distribution at the mRNA and protein level is differentially modulated in P2X7R deficient mice days 2-30 post-IR. Unexpectedly, the most well characterized EP-mediated signaling pathways, measured by phosphorylation of CREB and Akt are minimally different between genotypes days 2-30 post-IR and CREB-activated gene targets are inconsistently modulated across timepoints. Likewise, phosphorylation of p38 MAPK and ERK1/2 are modestly different across timepoints. Interestingly, phosphorylation of JNKs and their downstream target, c-Jun are different between genotypes days 7-30 post-IR and may explain the differences in salivary gland function seen between strains. Shockingly, evaluation of these pathways in primary cell cultures generated different results, indicating other cell types, such as immune cells, are likely influencing radiation-activated signaling pathways in vivo and should be further evaluated. JNK/c-Jun pathway activation is known to occur following radiation and influences the compensatory proliferation response. Targeting PGE2 production to modulate JNK pathway activation may provide novel therapeutic options to improve salivary gland function post-IR. Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that functions by inhibiting cyclooxygenase (COX) enzymes to block eicosanoid production. Data within this dissertation implicated PGE2 as a critical signaling mediator leading to radiation-induced salivary gland dysfunction, with activation of the JNK/c-Jun pathway correlating with the observed loss of function seen in wildtype mice. To further evaluate the role of PGE2-JNK-c-Jun signaling in radiation damaged salivary glands, mice were exposed to radiation and received injections of indomethacin with different dosing regimens. Remarkably, mice receiving three injections of indomethacin after radiation exposure (days 3, 5, and 7) have improved salivary gland function at day 30 post-IR. Indomethacin treatment to primary parotid cells reduced COX-1 activity and decreased PGE2 secretion. PGE2 treatment to primary parotid gland cells led to phosphorylation of JNK and c-Jun. PGE2 also induced cell proliferation, decreased amylase levels and reduced store operated calcium entry in cells following carbachol stimulation. Importantly, indomethacin treatment to mice decreased JNK pathway activation and rescued amylase levels in whole parotid tissues following radiation. Combined, these data support the notion that PGE2 production days 3-7 post-IR is detrimental to salivary glands due to JNK-c-Jun pathway activation and modulating this pathway via indomethacin treatment may be a promising therapeutic for restoring salivary gland function following radiotherapy.