Mycobacterium bovis bacille Calmette-Guérin (BCG) enhances human beta-defensin-1 gene transcription in human pulmonary gland epithelial cells.
Abstract
AIM: To examine the stimulatory effect of bacille Calmette-Gu rin (BCG) cell wall components on human beta-defensin-1 (hBD-1) gene expression and analyze the response element in the 5'-flanking region of the gene.
METHODS: BCG cell wall proteins were fractionated by Sephadex G-150 chromatography. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern hybridization analysis, hBD-1 mRNA expression was detected in a human pulmonary gland epithelial cell line SPC-A-1 cells. Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into promoterless chloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT reporter plasmids. Reporter gene expression was determined by ELISA.
RESULTS: There was an obvious enhancement of hBD-1 mRNA expression after stimulation with heat-inactivated BCG whole cells (50 mg/L), or the cell wall components with a molecular weight of 18-30 kDa (3 mg/L) for 8 h. The upstream sequence between -314 bp and +54 bp had the inducible activity by BCG, which contained CCAAT/enhancer binding protein-beta (C/EBP beta), activator protein-1 (AP-1), and CP2 cis element.
CONCLUSION: BCG cell wall components (18-30 kDa) can stimulate hBD-1 mRNA expression in pulmonary gland epithelial cells. The sequence (-314/+54) containing C/EBP beta, AP-1, and CP2 binding sites in the upstream of hBD-1 is involved in this induction.
Keywords:
METHODS: BCG cell wall proteins were fractionated by Sephadex G-150 chromatography. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern hybridization analysis, hBD-1 mRNA expression was detected in a human pulmonary gland epithelial cell line SPC-A-1 cells. Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into promoterless chloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT reporter plasmids. Reporter gene expression was determined by ELISA.
RESULTS: There was an obvious enhancement of hBD-1 mRNA expression after stimulation with heat-inactivated BCG whole cells (50 mg/L), or the cell wall components with a molecular weight of 18-30 kDa (3 mg/L) for 8 h. The upstream sequence between -314 bp and +54 bp had the inducible activity by BCG, which contained CCAAT/enhancer binding protein-beta (C/EBP beta), activator protein-1 (AP-1), and CP2 cis element.
CONCLUSION: BCG cell wall components (18-30 kDa) can stimulate hBD-1 mRNA expression in pulmonary gland epithelial cells. The sequence (-314/+54) containing C/EBP beta, AP-1, and CP2 binding sites in the upstream of hBD-1 is involved in this induction.