Differential autophagic cell death under stress with ectopic cytoplasmic and mitochondrial-specific PPP2R2B in human neuroblastoma cells

Abstract

Protein phosphatase 2A is one of four major classes of serine/threonine phosphatases. Overexpression of brain-specific regulatory subunit PPP2R2 in neuron cells is implicated in pathogenesis. The alternative splicing of PPP2R2B encodes two isoforms. They are subunit of cytoplasmic specific Bβ1 and mitochondria-targeted Bβ2. The two constructs were transfected into human neuroblastoma cells, SK-N-SH, respectively, and the stable clones overexpressing either Bβ1 or Bβ2 established. We have reported that Bβ2 clones are sensitive to reactive oxygen species (ROS) treatment by inducing autophagic cell death. To study more on the onset of neuropathogenesis under strain, both clones were exposed to different environmental stress, e.g. starvation and endoplasmic reticulum (ER) stress. To learn how PPP2R2B overexpression responds to starvation, cells were incubated in Hank's buffered salt solution of deprived nutrient. Cell death was induced in Bβ1 clones after 6 h starvation, but not in Bβ2 clones. The pharmacological inhibitor, Bafilomycin A1, rescued the cell death while suppressing autophagy. On the other hand, to assess how cells respond to ER stress, the cells were treated with 0.1 μM of N-glycosylation inhibitor, tunicamycin (TM). In contrast with Bβ1, the apoptotic cell death appeared in Bβ2 after 48 h treatment. The formation of autophagolysosome was detected in Bβ2 following 12 h treatment with TM as evidenced by lysotracker and GFP-LC3 staining for fluorescence microscopy analysis. The autophagy inhibitor, 3-methyladenine, salvaged the final apoptosis. The stable cell lines with ectopically transfected PPP2R2B genes encoding isoforms of brain-specific regulatory subunit exhibit distinct apoptosis under different stressors. The induced autophagic apoptotic cell death is related to mitochondrial membrane potential drop and ROS generation. Disturbance of autophagy alleviates the induced cell death. The results promised a good model for understanding the onset in pathogenesis under stress in neuron cells with aberrant PPP2R2B expression.

Energy crisis: The role of oxidative phosphorylation in acute inflammation and sepsis

“…PP2A is a multifunctional heterotrimeric serine/threonine protein phosphatase composed of a scaffolding A subunit, a catalytic C subunit, and a regulatory B subunit, the latter of which is expressed as several distinct isoforms. For example, among B subunit isoforms, the brain-specific splice isoform Bβ2, upon neurotoxic stress, translocates from the cytosol to the outer mitochondrial membrane, triggered by phosphorylation of three N-terminal residues [85], to induce mitochondrial fission and to execute apoptosis [86], a process which is accompanied by increased ROS production [87]. Mitochondrial PP2A also provides a direct link to autophagy, which is induced in several disorders including neurodegenerative disease due to mitochondrial dysfunction or cellular stress and can further enhance cellular demise [88].…”

Synergistic anti‐tumour effects of tetrandrine and chloroquine combination therapy in human cancer: a potential antagonistic role for p21

“…Although autophagy is a survival mechanism, it has also been suggested that autophagy could be cytotoxic when cells suffer treatment‐induced stress. Excessive cell damage will lead to autophagic cell death (Fang et al ., ; Li et al ., ). In the present study, we have investigated, in cancer cells the effects of tetrandrine‐induced stress in the presence of chloroquine, which prevents autophagy by blocking autophagosome fusion and degradation.…”