title: The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA.
abstract: BACKGROUND
  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) play an important role in the development of these diseases. Mitogen-activated protein kinase (MAPK) p38/activated protein kinase 2 (MK2) regulates the expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells (HPMECs) stimulated by tumor necrosis factor-α (TNF-α); however, the underlying molecular mechanism remains unclear. Here, we show that human antigen R (HuR), an RNA binding protein which binds preferentially to AU-rich elements (AREs) and stabilizes mRNAs, regulates TNF-α-induced ICAM-1 expression in the MK2/HuR signaling pathway.
  
  
  METHODS
  MK2 and HuR were silenced respectively in HPMECs and then HPMECs were stimulatied with TNF-α. Nucleo-cytoplasmic shuttling of HuR was detected by subcellular fractionation and confocal microscopy in MK2 knockdown HPMECs. In HuR silencing cells, protein and mRNA levels of ICAM-1 and IL-8 were measured by western blot analysis, ELISA and real-time PCR; mRNA stabilization were measured by real-time PCR after actinomycin D (ActD) blocking transcription. Furthermore, we performed neutrophil adhesion assay to assess the adhering capacity after HuR silencing.
  
  
  RESULTS
  MK2 were subjected to a knockdown by interfering RNA, the mRNA and protein levels of HuR in human pulmonary microvascular endothelial cells (HPMECs) were not affected. However, after the stimulation of TNF-α, silencing MK2 inhibited HuR accumulation to cytoplasm from nucleus in HPMECs. Consequently, knockdown of HuR by RNA interference in HPMECs, there was reduction in the stability of ICAM-1 mRNA and ICAM-1 protein level. This event was accompanied by a decrease in the adhesion of neutrophils towards HPMECs. Nevertheless, HuR silencing had no effect on the mRNA and protein levels of IL-8.
  
  
  CONCLUSIONS
  These results indicate that MK2 post-transcriptionally regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs.
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, 210002, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wu
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, 210002, China.|Department of Respiratory Medicine, Lianyungang First People's Hospital, Affiliated Hospital of Xuzhou Medical College, Clinical Medical School of Nanjing Medical University, Lianyungang, 222002, China.
authors.forename: Jia-Xin
authors.initials: JX
authors.lastname: Shi
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Southern Medical University, Guangdong, 510000, China.
authors.forename: Shen
authors.initials: S
authors.lastname: Geng
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, 210002, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, 210002, China. shiyi56@126.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Respiratory and Critical Care Medicine, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, 210002, China. suxinjs@163.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Su
mesh_terms: D002478:Cells, Cultured; D000067780:ELAV-Like Protein 1 / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D000067780:ELAV-Like Protein 1; D018799:Intercellular Adhesion Molecule-1; D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000099190:MAP-Kinase-Activated Kinase 2; C508606:CXCL8 protein, human; C098770:ELAVL1 protein, human
keywords: HPMEC; HuR; ICAM-1; IL-8; MK2