Effect Of Hypoxia On The The Mitophagy And The Regulation Mediated By BNIP3

Hypoxia is a representative stress environment. Organism subjected to severehypoxia can result in many kinds of diseases, such as stroke, cerebral palsy. On theother hand, adaptive responses are induced to promote survival under moderatehypoxia. It will be conducive to understand the mechanisms of hypoxic adaptation ifcell metabolism under different hypoxia is revealed. When cells are exposed tohypoxic stress, amount of reactive oxygen species (ROS) are generated atmitochondrial electron transport chain (ETC), which attack cellular macromoleculesor organelles and subsequently lead to cell dysfunction or death.Mitophagy was recently found as an adaptive metabolic response to hypoxia.Autophagy has been viewed as a way of cell death in the past decades. However, itwas until to now researchers noticed that autophagy can promote survival under stressenvironments. As one of autophagy, mitophagy can selectively clear the damagedmitochondria by autophagolysosome pathway and therefore maintain the ROS level,prevent apoptosis or necrosis resulted from the damaged mitochondria. Such, it isconsidered that mitophagy plays an important role in promoting survival undervarious stress conditions. Recent investigation showed that mitophagy was induced bythe pathway mediated by HIF-1, BNIP3and PI3K, and then the generation of ROSwas blocked, finally cell survival was promoted. This process is beneficial to theorganism adapt to hypoxia.BNIP3is a member of BH3-only subfamily in Bcl-2family. It is a mitochondrialouter membrane protein. It was reported that BNIP3is a target gene of HIF-1. Earlystudies showed that BNIP3plays an important role in the mitochondrial pathway ofapoptosis and necrosis. In recent years, growing studies reported that BNIP3promotes survival by mitophagy. Therefore, there exist two opposing views about the functionsof BNIP3.What effects do different degrees of hypoxia on mitophagy, cell survival andBNIP3expression? How does hypoxia regulate BNIP3and miotphagy and furtheraffect cell survival? These questions are not elucidated yet.In this study, we used different degrees of hypoxia (10%O2and0.3%O2) to probethe relationship between cell suvivral and mitophagy, and to find out the expressionpatterns of BNIP3and its regulation on mitophagy under different hypoxia. This studywill increase the new contents in mitophagy research.The main results are as follows:1. Effects of different hypoxia on cell survival statusModerate hypoxia (10%O2,24h) and severe hypoxia (0.3%O2,24h) were usedto detect the effects of hypoxia on cell status. Through detection of cell viablity, totalanti-oxidant capacity (T-AOC), levels of ROS, cell apoptosis and proliferation, wecompared the effects of different hypoxia on cell survival status.1.1Cell viabilityWe compared the effects of different oxygen concentrations and differenthypoxia time on cells viability at different seeding densities. The results showed thatthe cell viability was increased after cells were exposed to10%O2for24h or48h,and decreased after exposed to0.3%O2for24h or48h.1.2Total anti-oxidant capacityWe furthermore detected the total anti-oxdiant capacity of PC12cells underdifferent hypoxia using commercial assay kits. The data demonstrated that the T-AOCwas increased under moderate hypoxia (10%O2,24h) and decreased under severehypoxia (0.3%O2,24h)1.3ROS contentTo measure the levels of ROS, we incubated PC12cells with the fluorescenceprobe. Compared to normoxia, moderate hypoxia did not affect the fluorescencedensity, which suggests that ROS contents were unchangeable; severe hypoxiaenhanced the fluorescence density by2-fold, which suggests that ROS contents wereincreased approximately by2-fold. 1.4Cell apoptosisIn view of the excessive ROS generation is one of mechanisms of apoptosis. Weanalyzed cell apoptosis by TUNEL staining, Annexin V/PI double-staining and theexpressions of active forms of Caspase-3and Caspase-9under different hypoxia.Compared to normoxia, moderate hypoxia did not affect apoptosis, while severehypoxia significantly elevated apoptosis.1.5Cell proliferationThe number of cell survival was not only affected by apoptosis, also byproliferation.So, we further detected the cell proliferation by PI staining and BrdUimmunostaining. The cell proliferation was not affect by moderate hypoxia, butinhibited by severe hypoxia.In short, through the above studies, we determined two different hypoxia models:moderate hypoxia (10%O2,24h) and severe hypoxia (0.3%O2,24h). And we gotsuch a conclusion that cell survival status was different under different hypoxia.2. Effects of different hypoxia on mitophagy and the roles of mitophagy in cellsurvival statusMitophagy was recently found as an adaptive metabolic response to hypoxia.How does hypoxia regulate miotphagy and further affect cell survival? To clarifythese questions, we carried out the following experiments:2.1Mitophagy under different hypoxiaAfter PC12cells were exposed to20%O2,10%O2or0.3%O2for24h, weanalyzed mitophagy by electron microscopy, western blot, and fluorescencecolocalization. Compared to normoxia, moderate hypoxia promoted mitophagy, andsevere hypoxia inhibited mitophagy.2.2The effect of inhibition of mitophagy on cell survival statusPC12cells were treated with autophagy inhibitor3-MA followed by exposure to20%O2,10%O2or0.3%O2for24h, then cell viability, ROS content and apoptosiswas dectected by MTT, ROS probe and TUNEL staining, respectively. The resultsshowed that cell viability was dercreased in normoxia and moderate hypoxia, whileunchanged in severe hypoxia after cells were pretreated with3-MA; at the same time,the levels of ROS and apoptosis were increased by3-MA in the three groups,especially in the moderate hypoxia group. These results indicate that moderate hypoxia induced mitophagy, reduced ROS and apoptosis, and ultimately promotedcell survival; while severe hypoxia led to the inhibition of mitophagy, then theexcessive amount of ROS, the increase of apoptosis, and ultimately the decrease ofcell survival.2.3Comparison of the roles of mitophagy and apoptosis in cell survival statusunder different hypoxia.In addition, by comparison of cell viability and apoptosis by MTT and TUNELstaining under different hypoxia after cells were treated with3-MA or Z-VAD(apoptosis inhibitor), we analyzed the changes of cell survival status, and drew apreliminary conclusion: under moderate hypoxia, mitophagy is the main regulatorypathway; under severe hypoxia, apoptosis is the main regulatory pathway.3. The effect of hypoxia on BNIP3expression and its regulation on mitophagy3.1The expression of BNIP3under different hypoxiaThe protein level of BNIP3was measured24h after cells were exposed tohypoxia. The results showed that in normoxia, BNIP3protein level was lower; inmoderate hypoxia, the protein level of BNIP3was sharply elevated; however, severehypoxia resulted in a significant drop in BNIP3protein level.Using real-time PCR and western blot assays, we tested the dynamic changes ofBNIP3mRNA and protein at different time-points under hypoxia. The data revealedthat under moderate hypoxia, the levels of BNIP3mRNA and protein increased withtime; Under severe hypoxia, BNIP3mRNA significantly elevated with time, whereasits protein level increased at first and then declined, which hints somepost-translational modification involved in the regulation of BNIP3protein level.3.2The impact of enforced over-expression of BNIP3on mitophagyPC12cells were transfected with a BNIP3plasmid containing a flag tag,followed by detecting the marker molecules of mitophagy by western blot. The resultsshowed that the enforced over-expression of BNIP3promoted the induction ofmitophagy.3.3The impact of BNIP3knockdown on cell survival, apoptosis and mitophagyPC12cells were transfected with BNIP3siRNA, and then cell survival, apoptosisand mitophagy was measured by MTT, western blot and TUNEL assays, respectively.As we expected, the BNIP3knockdown reversed the effects of moderate hypoxia on mitophagy, apoptosis, and cell survival; however, it did not obviously changed theeffects of severe hypoxia on mitophagy, apoptosis, and cell survival.Taken together, this study demonstrated that moderate hypoxia (10%O2)promoted cell survival through removal of excessive ROS by mitophagy, and thenmaintaining the redox balance and reduction of apoptosis; severe hypoxia (0.3%O2)led to the inhibition of mitophagy, and then accumulation of excessive ROS,imbalance of cellular redox and increase of apoptosis. We first proved that thedifferences in BNIP3protein expression mediated the cell survival under differenthypoxia. This study analyzed the relationship between cellular metabolism and cellsurvival from a new perspective of mitophagy mediated by BNIP3under differenthypoxia, and added new content for mitophagy field.