The Role Of PKM2 In The Inflammatory Response Of Microglia After Brain Ischemia

Ischemic cerebrovascular disease (ICVD) is a kind of disease which is mainly caused by intracranial vascular lesions, which contains transient ischemic attack, cerebral infarction and moyamoya disease. The pathogenesis of brain ischemia is multifactorial, including oxidative stress, the toxicity of excitatory amino acids, the overload of intracellular Ca2+, inflammatory response and so on. Among them, inflammatory response which is induced by microglia cells plays an important role in neurotoxicity of brain ischemia.The pyruvate kinase isoenzyme M2 (PKM2) is a key enzyme of glycolysis which catalyzes the last step, the dephosphorylation of phosphoenolpyruvate converting to pyruvate and is responsible for the ATP production within the glycolytic sequence. PKM2 is present in embryonic and neoplastic tissue. So far, studies on PKM2 are around cancer cells and the switch to the PKM2 isoform, observed in tumors of different origin was shown to be essential for both the "Warburg" effect and proliferation of cancer cells. The Warburg effect, a switch from aerobic energy production to anaerobic glycolysis, promotes tumor proliferation and motility by inducing acidification of the tumor microenvironment. Although aerobic glycolysis (the Warburg effect) is a hallmark of cancer cells, we hypothesized that whether PKM2 is fuctional in hypoxia microglia cells since the oxygen-loss environment is similar to tumor cells.Our studies were designed to explore the effect and mechanism of PKM2 in brain ischemia. Firstly, We set up the middle cerebral artery occlusion (MCAO) model in rat and detected the expression of PKM2 after brain ischemia. The results of immunohistochemical staining showed that PKM2 was expressed in microglia and the expression was increased after ischemia. This result provided us a foundation for in vitro study. In vitro study, We used BV-2 cell line to set up a hypoxic microglia cell model by incubating the cells in serum-free medium with 250μM andcobalt chloride hexahydrate (CoCl2) or in a chamber filled with a gas mixture of 1% O2,5% CO2 and 94% N2. The results of Real-time polymerase chain reaction and western blot analysis showed that the expression of both PKM2 and hypoxia-inducible factor-1α (HIF-1α) were obviously increased under these conditions compared to control group. These results confirmed our hypothesis that PKM2 plays an important role in hypoxic microglia cells. Further studies revealed that both PKM2 and HIF-1α protein levels were significantly increased in hypoxia BV-2 nucleus but not in cytoplasm compared with control group. These results showed that the increase of PKM2 level may depend on its protein kinase activity in the nucleus. Additionally, we proved that the increase of PKM2 was related to HIF-1α directly. Firstly, we found that the mRNA level of PKM2 was reduced in BV-2 cells which were transfected with HIF-1α siRNA while the mRNA level of PKM2 was increased in BV-2 cells which were transfected with HIF-1α expression plasmid. Then we performed chromatin immunoprecipitation (ChIP) assays with HIF-1α monoclonal antibody. Using specific primer of mouse PKM2 HRE to amplify the DNA which was purified from hypoxia-treated BV-2 microglia cells, we further found that hypoxia induced the binding of HIF-1α protein together with PKM2 HRE in BV-2 cells. To test whether this binding is functional, we designed a reporter plasmid which contains a HRE fragment inserted to the downstream of firefly luciferase (FLuc) coding sequences. HEK-293T cells were first transfected with HRE reporter plasmid. The Fluc activity was significantly increased in hypoxic HEK-293T cells after inducing by 250μM CoCl2. Further more, we found that the Fluc activity was reduced after cotransfecting with both HIF-1α siRNA and pGL3-HRE reporter comprared with the corresponding control siRNA and pGL3-HRE reporter in HEK-293T cells. Taken together, these data indicated that HIF-1α binds to an HRE within the intron of PKM2 and directly activates PKM2 transcription in hypoxia microglia.Studies confirmed that in human lung cancer cells, acute increases of intracellular reactive oxygen species (ROS) caused inhibition of PKM2 through oxidation of Cys358 to generate sufficient reducing potential for detoxification of ROS. In hypoxic BV-2 cells, we observed the increase of ROS production and some inflammation mediators (IL-1β, IL-6, IL-18 and VCAM-1) contrast to control group. The mRNA levels of these mediators increased significantly in BV-2 cells transfected with PKM2 expression plasmid. Interestingly, this increase could be attenuated by the mutant on Cys358 to Ser358. Therefore, we reach a conclusion that PKM2 plays a role in microglia inflammation through the oxidation of its Cys358 site.Above all, the increase of PKM2 level is regulated by HIF-la directly under hypoxic-ischemic stimulus in BV-2 microglia cells, which causes the transcription of inflammatory mediators such as IL-1β, IL-6, IL-18 and VCAM-1, finally results in the inflammatory response.Our report illustrated the function of PKM2 in the inflammatory response of microglia after hypoxic-ischemic injury, providing a potential therapeutic target to reduce brain ischimic injury.