The Mechanism Of Pak4 In Maintaining The Pluripotency Of Mouse Embryonic Stem Cells

Embryonic stem cells(ESCs)have the ability to stay self-renewal in vitro and the pluripotency to develop into an entire individual.Exploiting the pluripotent properties of ESCs hold great promise for regenerative medicine.However,there are technical limitations and ethical issues in investigation of human ESCs.In this case,it is quite important to extent the application of mouse ESCs(mESCs)in medicine and clinical research and to understand the mechanism of how the pluripotency maintaining.Nevertheless,directing ESCs differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks.The p21-activated kinase 4(Pak4),a serine/threonine kinase belonging to group Ⅱ p21-activated kinase family members,is previous reported to be associated with mouse embryonic development.Pak4 knockout mice has embryos lethality with defects in fetal heart.Pak4-deficient in nervous system leds to dramatic defects in neuronal development.Based on those background,we focused on Pak4 protein in sustaining mESCs pluripotency in our research.According to our study,we figured out that Pak4 is upregulated in mESCs compare with MEF cells.When treated with RA or during embryonic body formation process,the protein level of Pak4 in embryonic stem cells is down-regulated and mESCs can not maintain pluripotency.Knockdown of Pak4 in mESCs leads to downregulation of pluripotency markers and decreased AP positive clone numbers.Consistent with this,mESCs could maintain pluripotency when cultured in LIF-free N2B27 medium with overexpression of Pak4.Meanwhile,we found that Pak4 can improve somatic reprogramming efficiency.During MEF cells reprogramming,the AP positive colony number of iPS cells is higher when combine Pak4 with Yamanaka factors(Oct4/Sox2/Klf4/c-Myc)than that only use Yamanaka factors.On the other hand,the colony number of iPS cells is lower when Pak4 knockdown even transfection with Yamanaka factors.Furthermore,those iPS cells,which derived from Pak4 and Yamanaka factors transduced MEF cells,can express pluripotency marker proteins,such as Nanog,Fbx15 and SSEA1.Those iPS cells can also get teratomas contained various tissues derived from three germ layers through injecting of those iPS cells to nude mice.However,the ability of teratomas formation is largely weakened in Pak4 knockdown iPS cells.To investigate the mechanism of Pak4 in maintaining the pluripotency of mESCs.We first explore why Pak4 expression level is higher in mESCs and iPSCs compare with MEF cells.We predicted the transcriptional factor of Pak4 gene through JASPAR website and identified a putative binding site of Nanog at the promoter region of Pak4 gene.Then,we verified it through dual-luciferase reporter assay system and ChIP assay.Pak4 is transcriptionally regulated by core transcription factor Nanog.Moreover,we found that Pak4 could change the phosphorylation of Akt,an important kinase of signaling transmission in the maintaining of mESCs pluripotency.Through immuniprecipitation assay,we found that Pak4 could directly interact with and phosphorylate Akt.The loss-function mutant of Akt at serine 473 and threonine 308 would impair the interaction between Pak4 and Akt.Futher in vitro phosphorylation assay demonstrated that Pak4 could phosphorylate Akt at serine 473.The phosphorylation inhibition of Akt by MK2206 could impair the function of Pak4 in the maintaining of stemness.The constitutive activition of Akt could partially rescue the differentiation caused by Pak4 knockdown.Above all,the mechanism of Pak4 in maintaining the pluripotency of mouse embryonic stem cells is described below.Pak4 is transcriptionally regulated by Nanog and participates partly in the stemness maintaining function of Nanog as a transcriptional target.Meanwhile,Pak4 interacts with and phosphorylates Akt at serine 473,thereafter leading to the activation of Akt signaling and promoting the stemness of mESCs.