The Absence Of Pyruvate Kinase Induces Cell Senescence

Defined as stable cell-cycle arrest,cellular senescence plays an important role in diverse biological processes including tumorigenesis,organismal aging,and embryonic development.Cellular senescence is a complex process,and the factors involved in the regulation are diverse which interact with each other,constituting a large and fine regulatory network.In recent years,many studies have confirmed that the senescent cells have undergone significant metabolic changes,whereas the intermediate products of cell metabolism play an important role in cell senescence.We studied the last step of glycolysis:Phosphoenolpyruvate(PEP)converted to pyruvate.There is a key rate-limiting metablic enzyme,pyruvate kinase(PK).In human fibroblast(HFF),there are two types of pyruvate kinase isoforms-PKM1 and PKM2.The absence of pyruvate kinase(M1 and M2)can cause senescence of human fibroblast,including cells growth arrest,more SA-P-Gal staining positive cells,G2/M cell cycle arrest,high cellular respiration and senescence-associated secretory phenotype(SASP)factors secretion.PKM knockdown activated DNA damage response(DDR)and p53-p21 pathway.Total reactive oxygen species(ROS)didn't show any differences but mitochondria ROS had a significant upregulation compared to control.The absence of PKM made mitochondria hyperactive function and higher mitochondria ROS,activing DDR and p53-p21 pathway which induced cell cycle arrest.Unlike human fibroblasts(the terminal differentiation cells),stem cells' feature is pluripotency.So in human embryonic stem cells(hESC),PKM may have new functions which is different from one in HFF.Knock-down PKM hESC had S cell cycle arrest and weak colony forming ability,activing DDR like data about HFF.One of pluripotent genes—Nanog showed higher RNA expression but lower protein level.All those evidence suggested PKM allows hESC to remain pluripotent.To further illustrate the role of pyruvate kinase in vivo about aging,we chose the excellent model animal--caenorhabditis elegans.There are two types of pyruvate kinase genes,PYK1 and PYK2 on different chromosomes,and the coding sequence is approximately 50%similar.Using CRISPR/Cas9,we established PYK1::GFP and PYK2::GFP to expound the gene expression pattern.PYK1 showed expression in embryo,body wall of larva stage,and for adult elegans additional reproductive system.This implied PYK1 plays a key role in embryo and larva development.PYK2 mainly expressed in intestine for larva and adult.According to entirely different expression pattern,we focused on PYK1 to research pyruvate kinase function during aging.PYK1 KnockOut strain had serious phenotypes,such as embryonic lethal and larva dysplasia.Although PYK1 KnockOut strain adult showed smaller body size,we couldn't attribute this change to accelerated aging because it may be induced by defects in embryo and larva.We needed a more appropriate method to probe PYK1' role in elegans aging.