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).
Conclusion: 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.