Cellular and Molecular Neurobiology (2019) 39:241–254 https://doi.org/10.1007/s10571-018-0644-z ORIGINAL RESEARCH Retinoic Acid-Induced Protein 14 (RAI14) Promotes mTOR-Mediated Inflammation Under Inflammatory Stress and Chemical Hypoxia in a U87 Glioblastoma Cell Line XiaoGang Shen1,2 · JiaRui Zhang1,2 · XiaoLong Zhang1,2 · YiFan Wang1,2 · YunFeng Hu1,2 · Jun Guo1,2,3 Received: 25 August 2018 / Accepted: 8 December 2018 / Published online: 15 December 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Retinoic acid-induced 14 is a developmentally regulated gene induced by retinoic acid and is closely associated with NIK/ NF-κB signaling. In the present study, we examined the effect of RAI14 on mTOR-mediated glial inflammation in response to inflammatory factors and chemical ischemia. A U87 cell model of LPS- and TNF-α-induced inflammation was used to investigate the role of RAI14 in glial inflammation. U87 cells were treated with siR-RAI14 or everolimus to detect the correlation between mTOR, RAI14, and NF-κB. CoCl2-stimulated U87 cells were used to analyze the effect of RAI14 on mTOR-mediated NF-κB inflammatory signaling under chemical hypoxia. LPS and TNF-α stimulation resulted in the upregulation of RAI14 mRNA and protein levels in a dose- and time-dependent manner. RAI14 knockdown significantly attenuated the level of pro-inflammatory cytokine via inhibiting the IKK/NF-κB pathway. Treatment with an mTOR inhibitor (everolimus) ameliorated NF-κB activity and IKKα/β phosphorylation via RAI14 signaling. Notably, RAI14 also enhanced mTOR-mediated NF-κB activation under conditions of chemical hypoxia. These findings provide significant insight into the role of RAI14 in mTOR-induced glial inflammation, which is closely associated with infection and ischemia stimuli. Thus, RAI14 may be a potential drug target for the treatment of inflammatory diseases. Keywords RAI14 · Neuroinflmamation · mTOR · NF-κB · Chemical hypoxia Abbreviations 4EBP1 Eukaryotic initiation factor 4E binding protein1 CNS Central nervous system CoCl2 Cobalt chloride XiaoGang Shen and JiaRui Zhang have contributed equally to this work. Co-IP Co-immunoprecipitation eIF4E Eukaryotic translational initiation factor 4 Electronic supplementary material The online version of this epsilon article (https ://doi.org/10.1007/s1057 1-018-0644-z) contains Evero Everolimus supplementary material, which is available to authorized users. * Jun Guo 1 State Key Laboratory Cultivation Base For TCM Quality [email protected] and Efficacy, School of Medicine and Life Science, Nanjing University of Chinese Medicine, Nanjing 210023, XiaoGang Shen People’s Republic of China [email protected] 2 Key Laboratory of Drug Target and Drug for Degenerative JiaRui Zhang Disease, Nanjing University of Chinese Medicine, [email protected] Nanjing 210023, People’s Republic of China XiaoLong Zhang 3 Department of Biochemistry and Molecular Biology, [email protected] Jiangsu Key Laboratory of Therapeutic Material of Chinese YiFan Wang Medicine, School of Medicine and Life Science, Nanjing [email protected] University of Chinese Medicine, Nanjing 210023, People’s Republic of China YunFeng Hu [email protected] Vol.:(0123456789)1 3 242 Cellular and Molecular Neurobiology (2019) 39:241–254 IκB-α NF-kappa-B inhibitor alpha the activation of the mTOR/IκB-α/NF-κB pathway (Temiz- IKK Nuclear factor NF-kappa-B inhibitor kinase Resitoglu et al. 2017). Moreover, the mammalian target of LPS Lipopolysaccharide rapamycin (mTOR) functions as a serine/threonine pro- mRNA Messenger RNA tein kinase and controls protein synthesis, contributing mTOR Mammalian target of rapamycin to the associated physiological and pathological outcome mTORC Mammalian target of rapamycin complex (Caccamo et al. 2018; Wendel et al. 2004). mTOR is com- NF-κB Nuclear factor kappa-light-chain-enhancer of monly comprised of mTOR complexes 1 (mTORC1) and activated B cells 2 (mTORC2). In addition, mTORC1 activity is primarily NIK NF-kappa-B-inducing kinase inhibited by everolimus (RAD001) (Han et al. 2016; Yun PAMPs Pathogen-associated molecular patterns et al. 2016), which has been shown to effectively treat can- PBS Phosphate-buffered saline cers and autoimmune diseases (Kingwell 2013; Vitiello et al. RAI14 Retinoic acid-induced protein 14 2015). Nevertheless, the precise molecular mechanisms Raptor Regulatory-associated protein of mTOR underlying mTORC1-mediated inflammatory stress have RT-qPCR Quantitative real-time polymerase chain yet to be sufficiently identified. reaction In the present study, we have explored the mechanisms si-NC Control siRNA. underlying the RAI14 in mTOR-mediated inflammation siR-RAI14 RAI14 siRNA using models of inflammation and chemical hypoxia. siRNA Small interfering RNA TLRs Toll-like receptors TNF-α Tumor-necrosis factor-α Methods Cell Culture and Treatment Introduction The human glioma cell (U87) or human umbilical vein The human novel retinal pigment epithelial cell gene (NOR- endothelial cell (HUVEC) was obtained from the American PEG, RAI14) is a developmentally regulated gene originally Type Culture Collection (ATCC, Manassas, VA, USA). U87 derived from the human retinal pigment epithelial cell line, and HUVEC cells were cultured in Dulbecco’s Modification ARPE-19 (Kutty et al. 2001). This gene encodes the RAI14 of Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) protein that contains six ankyrin repeats and an extended supplemented with 10% (v/v) fetal bovine serum (Gibco), coiled-coil domain. Moreover, RAI14 is widely expressed 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). in a variety of human tissues and participates in the reor- The cells were cultured in 60-mm culture dishes at 37 °C in a ganization of actin filaments in the testis, contributing to humidified atmosphere of 5%CO 2. U87 cells were grown on spermatid polarity and cell adhesion (Chen et al. 2018a, b; 60-mm culture dishes then incubated with 20 nM everolimus Peng et al. 2000; Qian et al. 2013). While the Gene Ontol- (MedChem Express, Monmouth Junction, NJ, USA) with ogy has deduced that RAI14 is associated with NIK/NF-κB or without TNF-α, LPS, or CoCl 2 as a baseline for further signaling (Huttlin et al. 2017), the function of RAI14 in glial research. Recombinant human TNF-α was obtained from inflammation remains unknown. Hangzhou Biologic Biotechnology (China). All cell experi- It has been well established that the NF-κB signaling ments were repeated at least three times. pathway is involved in the wide variety of pathological conditions, such as cancer and neurodegenerative diseases Antibodies (Collignon et al. 2018; Li et al. 2012). Under different patho- logical conditions (e.g., LPS, TNF-α and hypoxia), NF-κB Rabbit anti-RAI14 polyclonal antibody, rabbit anti-GAPDH p65 activation and nuclear translocation control various tran- polyclonal antibody, mouse anti-α-tubulin monoclonal anti- scriptional responses (Li et al. 2016). Thus, pro-inflamma- body, rabbit anti-phospho-mTOR (Ser2448) antibody and tory agonists, such as LPS and TNF-α, could play a vital role rabbit anti-phospho-eIF4EBP1 (Thr37 + Thr46) antibody in the progression of diseases of the central nervous system. were purchased from Bioss (Beijing, China). Phospho- Additionally, since hypoxia can trigger a pro-inflammatory IKKα/β (Ser176/180) antibody and NF-κB p65 rabbit mon- immune response (Cosin-Roger et al. 2017; Subhan et al. oclonal antibody were obtained from Beyotime (Shanghai, 2017), CoCl2 (cobalt chloride), a hypoxia-mimetic agent, China). Rabbit anti-α-actin monoclonal antibody, rabbit can be used to make an analogical analysis of the NF-κB anti-β-actin polyclonal antibody, and rabbit anti-β-tubulin inflammatory signaling pathway under conditions of chemi- monoclonal antibody were obtained from BOSTER (Wuhan, cal hypoxia. It has previously been shown that mTOR is China). All secondary antibodies were purchased from associated with proinflammatory immune responses through ZSGB-BIO (Beijing, China). 1 3 Cellular and Molecular Neurobiology (2019) 39:241–254 243 siRNA Transfection was evaluated using an ECL Western Bot analysis kit (Mil- lipore) and were normalized to those of α-actin or β-actin or The siRNA probe s25049 for RAI14, siRNA probe s25048 α-tubulin or β-tubulin or GAPDH. for RAI14 (Hasson et al. 2013), GAPDH positive control oligonucleotides and its control small interfering RNA Co‑immunoprecipitation (siRNA) in experiments refer to an All-Starnon silencing siRNA synthesized by Shanghai GenePharma (Shanghai, According to the manufacturer’s instructions, 2 µg NF-κB China). Lipo6000™ Transfection reagent (Beyotime) was p65 rabbit monoclonal antibody was added to the same used for the transfection of siRNA according to the manu- amount (500 µg) of cell lysate overnight at 4 °C. Protein facturer’s instructions. A/G-agarose spheres (Santa Cruz Biotechnology, Dallas, TX, USA) were added and the samples stored at 4 °C for 2 h. The samples were then centrifuged at 13,000g at 4 °C for Quantitative Real‑Time Polymerase Chain Reaction 1 min, washed three times with lysis buffer, and boiled for 10 min after the addition of 30 µL 1× SDS Loading Buffer. A Reverse Transcription Kit and the SYBR Green PCR Mas- The denatured samples were kept at − 20 °C for future west- ter Mix were obtained from Takara (Otsu, Shiga, Japan). ern blotting. Total RNA was isolated from the cells using Trizol reagent according to the manufacturer’s instructions (Beyotime) Immunofluorescence Analysis and cDNA was reverse-transcribed using a Reverse