Total RNA was used in the RT reactions using a SuperScript III Reverse Transcriptase kit according to the manufacturers instructions to synthesize the cDNA. The human PlGF and glyceraldehyde 3 phosphate dehydrogenase cDNA fragments were amplified from the cDNA by PCR, performed with Dream Taq DNA polymerase as follows now 5 min at 95 C, then 30 sec at 98 C, 30 sec at 59 C, and 1 min at 72 C for 35 cycles. The primer sets for mouse PlGF and GAPDH was as previously described. Chromatin immuno precipitation Genomic DNA fragment from BEAS 2B were prepared by the EZ Zyme Chromatin Prep Kit and analyzed using the Chromatin immuno precipitation Assay Kit to evaluate the associated levels of Egr 1 and PlGF promoter regions. Statistical analysis The results were presented as mean SEM from five independent e periments and animals.

The Mann Whitney test was used to compare two independent groups. Kruskal Wallis with Bonferroni post hoc analysis was used for multiple testing. Statistical analyses were performed using the SPSS version 8. 0. Statistical significance was set at p 0. 05. Results NE increased PlGF promoter activity by Egr 1 in LE Cells The results revealed that treatment with 100 300 mU ml NE for 24 h significantly increased PlGF promoter activity dose dependently in human bronchial epithelial cells, BEAS 2B, and primary mouse type II alveolar epithelial cell. Previous studies indicated that several conserved metal response elements and hypo ia response elements reside in mouse or human PlGF promoter regions.

However, treatment with 300 mU ml NE did not alter the e pression of mental regulatory transcription factor 1 and hypo ia inducible factor 1. Egr 1, and B actin were analyzed by Western blot analysis. The association of Egr 1 and PlGF promoter fragment was evaluated by chromatin immuno precipitation assay. Data are presented as mean SEM. p 0. 05 vs. vehicle treated group. There was a conserved Egr 1 response element in the human and mouse PlGF promoter regions near the transcriptional start site. Western blotting revealed that 300 mU ml NE challenge transiently increased Egr 1 e pression in BEAS 2B. By ChIP, treatment of 300 mU ml NE for 1 h triggered the binding of Egr 1 and PlGF promoter fragments in BEAS 2B and pre treatment with Egr 1 siRNA inhibited the NE increased PlGF promoter activity in BEAS 2B and AEC II. Thus, NE increased PlGF Batimastat promoter activity through the association of Egr 1 and the PlGF promoter fragment. NE increased PlGF e pression in LE Cells NE had been reported to up regulate elafin e pression in A549 cells and PlGF was majorly secreted by AEC II. To test whether NE could induce PlGF e pression, BEAS 2B and AECII were treated with of 0 300 mU ml NE for 24 h.