Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype

Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classically activated macrophage(M1)polarization.In this study,we established THP-1-derived testing state macrophages(M0),M1 macrophages,and alternately activated macrophages(M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins(CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated(PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence(IF)-RNA fluorescence in situ hybridization(FISH)double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.

Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein

AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analysis,sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells.Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR).Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins.In vivo 32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation,eIF2α phosphorylation,and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus.In a sucrose gradient,approxi-mately 30% of the overexpressed p67 was bound with ribosomes.p67 interacted with the kinase domain,butnot the dsRNA-binding domains of PKR.Only the glycosylated p67 was associated with the ribosome,and p67 did not compete with PKR for ribosome binding.In breast cancer cells,there was increased autophosphorylation of PKR but no phosphorylation of eIF2α,compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.

An ongoing search for potential targets and therapies for lethal sepsis

Sepsis, which refers to a systemic inflammatory response syndrome resulting from a microbial infection, represents the leading cause of death in intensive care units. The pathogenesis of sepsis remains poorly understood although it is attributable to dysregulated immune responses orchestrated by innate immune cells that sequentially release early(e.g., tumor necrosis factor(TNF), interleukin-1(IL-1), and interferon-γ(IFN-γ) and late(e.g., high mobility group box 1(HMGB1)) pro-inflammatory mediators. As a ubiquitous nuclear protein, HMGB1 can be passively released from pathologically damaged cells, thereby converging infection and injury on commonly dysregulated inflammatory responses. We review evidence that supports extracellular HMGB1 as a late mediator of inflammatory diseases and discuss the potential of several Chinese herbal components as HMGB1-targeting therapies. We propose that it is important to develop strategies for specifically attenuating injury-elicited inflammatory responses without compromising the infection-mediated innate immunity for the clinical management of sepsis and other inflammatory diseases.

DNA repair genes BRCA1 and DNA-PKcs have great potential in radiation therapy

Radiotherapy is a part of the front-line treatment regime for many cancers. The mechanisms of radiation-induced effects in cancers mainly involves double-strand breaks (DBS) which plays very important role in maintaining the stability of gene. As DNA repair gene breast cancer 1 (BRCA1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can act to maintain genetic stability though two distinct and complementary mechanisms for DNA DSB repair-homologous recombination (HR) and non-homologous end joining (NHEJ). Therefor, BRCA1 and DNA-PKcs are closely associated with radiation sensitivity, which means that they may be used as a useful tool to predict radio sensitivity in human tumour cells.

Endoplasmic reticulum stress and liver diseases

Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1 a(IRE1 a),double-stranded RNA-dependent protein kinase(PKR)-like ER kinase(PERK)and activating transcription factor 6(ATF6)signaling pathways,is a protective cellular response activated by ER stress.However,UPR activation can also induce cell death upon persistent ER stress.The liver is susceptible to ER stress given its synthetic and other biological functions.Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and the UPR signaling pathways in the pathogenesis of liver diseases,including non-alcoholic fatty liver disease(NAFLD),alcoholic liver disease(ALD),alpha-1 antitrypsin(AAT)deficiency(AATD),cholestatic liver disease,drug-induced liver injury,ischemia/reperfusion(I/R)injury,viral hepatitis and hepatocel-lular carcinoma(HCC).Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of the UPR pathways during the development of the diseases.Moreover,ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.