Figure 9.

The effect of site-directed mutation of the HSE region on heat-induced modulation of the occludin promoter activity and binding of HSF-1 to HSE on the occludin promoter. a: The 50-pb occludin promoter regions encoding either the wild-type HSE or mutated HSE motif were synthesized and used as a DNA probe to assess the binding of heat (41°C for 1 hour)-activated HSF-1 (nuclear fraction). HSF-1 binding was determined by ELISA-based DNA binding assay. Mutation of the HSE motif inhibited the heat-induced HSF-1 binding to the DNA probe. Data represent means ± SE (n = 3); *P < 0.05 versus 37°C; **P < 0.01 versus 41°C WT. b: The mutant occludin promoter containing the mutation of the HSE region was generated and then transfected into Caco-2 cells as described in the Materials and Methods. Transfected Caco-2 cells were treated with control temperature 37°C or heat 41°C for 2 hours. The mutation of HSE motif prevented the heat-induced increase in occludin promoter activity. The absolute values for the SEAP activity were 105,030 ± 8495, 118,108 ± 11,928, 174,903 ± 6738 and 118,937 ± 8158 for 37°C WT, 37°C Mut, 41°C WT, and 41°C Mut groups, respectively. Data represent means ± SE (n = 4 to 6); *P < 0.001 versus 37°C; **P < 0.001 versus 41°C WT. The experiments were repeated three times.