Phosphoinositide-3-kinase regulatory subunit 4 participates in the occurrence and development of amyotrophic lateral sclerosis by regulating autophagy

The development of amyotrophic lateral sclerosis(ALS)may be related to the abnormal alterations of multiple proteins.Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4(PIK3R4)was decreased in ALS.However,the role of PIK3R4 in ALS pathogenesis remains unknown.This study was the first to find that transfection of PC12 cells with small interfering RNA against the PIK3R4 gene significantly decreased the expression levels of PIK3R4 and the autophagy-related proteins p62 and LC3.Additionally,in vivo experiments revealed that the PIK3R4 protein was extensively expressed in the anterior horn,posterior horn,central canal,and areas surrounding the central canal in cervical,thoracic,and lumbar segments of the spinal cord in adult mice.PIK3R4 protein was mainly expressed in the neurons within the spinal lumbar segments.PIK3R4 and p62 expression levels were significantly decreased at both the pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1 Gur mice compared with control mice,but these proteins were markedly increased at the progression stage.LC3 protein expression did not change during progression of ALS.These findings suggest that PIK3R4 likely participates in the prevention of ALS progression.This study was approved by the Ethics Committee for Animal Care and Use of Jiangxi Provincial People’s Hospital,Affiliated People’s Hospital of Nanchang University(approval No.2020025)on March 26,2020.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

Intracellular and Extracellular Phosphatidylinositol 3-Phosphate Produced by Phytophthora Species Is Important for Infection

RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.

The Activation of Peroxisome Proliferator-activated ReceptorγEnhances Insulin Signaling Pathways Via Up-regulating Chemerin Expression in High Glucose Treated HTR-8/SVneo Cells

Objective:To investigate whether peroxisome proliferator-activated receptorγ(PPARγ)agonists,rosiglitazone and GW1929,activate the phosphatidylinositol 3-kinase(PI3K)-AKT/protein kinase B pathway and the mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase1/2(ERK1/2)pathway by upgrading the expression of chemerin.Methods:The HTR-8/SVneo trophoblastic cells were cultured in vitro in high glucose concentration(25 mmol/L)to mimic gestational diabetic phenotypes.We transfected small interfering RNA into HTR-8/SVneo cells to silence two receptors of chemerin,that are chemokine-like receptor 1(CMKLR1)and G protein-coupled receptor1(GPR1).And recombinant human chemerin,PPARγagonists(rosiglitazone,10μmol/L and GW1929,10μmol/L)and PPARγinhibitor(GW9662,5μmol/L)were additionally added to the medium,respectively.The existence of chemerin was verified by immunocytochemistry,and the expressions of PPARγ,chemerin,and its receptors as well as insulin signaling-related factors PI3K,AKT2,and MAPK(ERK1/2)were detected by real time quantitative-polymerase chain reaction and western blot.Results:Chemerin existed in the HTR-8/SVneo cells.Effects of chemerin on PI3K-AKT pathway and MAPK(ERK1/2)pathway were dependent on the density of chemerin.When rosiglitazone and GW1929 were added to the medium,the mRNA levels of PI3K,AKT2,and MAPK1 were upregulated(P<0.05).Conversely,GW9662 downregulated the mRNA levels of AKT2 and MAPK1(P<0.05).Rosiglitazone and GW1929 increased the protein levels of PPARγ,chemerin,CMKLR1 and GPR1(P<0.05).Rosiglitazone and GW1929 had no effect on the expression of PI3K p110βand phospho-AKT2 without CMKLR1(P>0.05).Meanwhile,the expression of phospho-ERK2 remained unaffected in the absence of GPR1(P>0.05).Conclusion:Both rosiglitazone and GW1929 have the effect of improving insulin signaling pathways via upgrading the level of chemerin in high glucose treated HTR-8/SVneo cells.