title: [Effect of osmotic response element binding protein on mucus secretion with hypertonicity in human airway epithelial cells].
abstract: OBJECTIVE
  To investigate the transcriptional regulation of osmotic response element binding protein (OREBP) to heat shock proteins (HSP)70 and its effect on mucus secretion under hypertonic conditions.
  
  
  METHODS
  Human bronchial epithelial HBE16 cells were cultured in vitro in hypertonic medium for different times. Western blot was used to analyze the levels of OREBP and HSP70-2 protein. The mucin(MUC)5AC protein content in supernatant were detected by enzyme linked immunosorbent assay (ELISA). Different deletions and mutants of HSP70-2 promoter in upstream region were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with OREBP siRNA and the promoter activities detected with dual luciferase assay to study the ORE site in the HSP70-2 promoter and its impact on MUC5AC.
  
  
  RESULTS
  Compared with control group (0.21 ± 0.05, 0.15 ± 0.06, 0.13 ± 0.04), the levels of OREBP, HSP70-2 and MUC5AC in supernatant significantly increased (0.54 ± 0.07, 0.20 ± 0.08, 0.17 ± 0.04) after HBE16 cells were exposed to 600 mOsm/L hypertonic mediums for 3 h (2.57, 1.33 and 1.31 fold in protein respectively) (all P < 0.05), and their expression contents increased in a time-dependent manner, for 6 h (2.81, 3.07 and 3.77 fold) (all P < 0.01), for 9 h (3.57, 5.13 and 4.00 fold) (all P < 0.01), for 12 h (4.24, 5.33 and 4.54 fold) (all P < 0.01). After a knock-down of OREBP by RNAi for 48 h, the levels of OREBP, HSP70-2 and MUC5AC significantly decreased (0.36 ± 0.08; 0.33 ± 0.08; 0.24 ± 0.05) versus the control group (0.95 ± 0.27, 0.75 ± 0.22, 0.58 ± 0.22) (protein inhibition ratio at 62%, 56% and 59% respectively) (P < 0.05, P < 0.01, P < 0.05). Luciferase assay indicated that an important ORE site in HSP70-2 promoter was in the region from -353 to -66. The inactivation of ORE site at -93 by site-directed mutagenesis led to a complete loss of HSP70-2 promoter activity (P < 0.01).
  
  
  CONCLUSIONS
  One ORE site at -93 in the HSP70-2 promoter and OREBP were found to play essential roles in inducing the HSP70-2 transcription and MUC5AC hypersecretion in human bronchial epithelial cells in response to hypertonicity.
authors.affiliation: Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400010, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Tu
authors.forename: Juliy M
authors.initials: JM
authors.lastname: Perelman
authors.forename: Victor P
authors.initials: VP
authors.lastname: Kolosov
authors.forename: Xiang-dong
authors.initials: XD
authors.lastname: Zhou
mesh_terms: D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050778:NFATC Transcription Factors / Q000378:metabolism*; D009995:Osmosis; D012137:Respiratory System / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D050778:NFATC Transcription Factors