Human β-defensin-1 Gene Expression And Transcription Regulation

Human β-defensin-1 (hBD-1), an important antimicrobial peptide defending against microorganism infection, was widely expressed in secretory glands and mucosal epithelial cells. Previously it was considered that the expression of hBD-1 was constitutive, and could not be enhanced by inflammation stimulator such as lipopolysaccharide (LPS) and TNF-a etc. However, recently it was reported that hBD-1 mRNA expression could be up-regulated by some microorganisms or inflammatory factors. Our group found that the expression could be obviously enhanced in pulmonary gland epithelial SPC-A-1 cells upon the stimulation with bacille Calmette-Guerin (BCG) cell wall components. Sequencing of the hBD-1 gene revealed that there were two exons and one intron spanning 6962 bp. NF- k B motif, which played an important role in regulating the inducible expression of some antimicrobial peptides such as human β -defensin-2 (hBD-2) by LPS, had not been found in the regulatory region of hBD-1 gene. However, investigation of hBD-1 gene promoter activity as well as its transcriptional regulation mechanism of the induction expression had not been systematically studied. Therefore we didsome works on that issue. There were three parts in this thesis: first, examining constitutive expression of hBD-1 mRNA in various cell lines; second, investigating the hBD-1 gene promoter activity; lastly, exploring the transcriptional regulation mechanism of the up-regulation of hBD-1 gene in SPC-A-1 cells upon the stimulation with BCG cell wall components.The gene expression of hBD-1 was investigated in several cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR) and northern hybridization. It was found that Pulmonary gland epithelial cell SPC-A-1, pulmonary epithelial cell A549, and mononuclear cell THP-1 constitutively expressed hBD-1 mRNA, while there was no hBD-1 mRNA signal to have been detected in the umbilical vein endothelium cell ECV304, fibroblast FB and 293 cells.In order to determine the promoter activity of hBD-1 gene, several upstream sequences of that gene were cloned into a promoterless plasmid pEGFP-1, and these constructs were transfected into SPC-A-1 cells respectively. The green fluorescence protein (GFP) expression was detected by fluorescence inversion microscope and flow cytometry. Results indicated that the promoter activity of -69 bp region was lower than that of -575 and -314 bp, both of which had powerful promoter activity.Furthermore, experiments were performed to examine the stimulatory effect of BCG cell wall components on hBD-1 gene expression and analyze the response element in the 5'-flanking region of that gene. Firstly, BCG cell wall proteins were extracted by sodium dodecyl sulfate (SDS) and fractionated by Sephadex G-150 chromatography. Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into another promoterlesschloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT hBD-1 reporter plasmids. One of them, -575 pCAT hBD-1 construct was applied to determine the effective components of BCG cell wall proteins which could enhance the expression of hBD-1. Results showed that the cell wall components with a molecular weight of 18 kDa to 30 kDa could enhance the transcription of hBD-1 gene. Secondly, SPC-A-1 cells were co-transfected with a series of pCAT hBD-1 constructs and pSV-P-Galactosidase control vector (as an internal control to normalize transfection efficiency) and stimulated with effective BCG cell wall components (3 mg/L) at the 6 hour after the transfection. Following incubation with BCG components for 39 hours, CAT and P-Gal protein expression were determined by ELISA. The concentration of CAT was normalized by that of P-Gal to indicate the transcription activity of the regulatory sequences. Results showed that the -314 bp region of hBD-1 gene was responsible for the up-regulated expression by BCG cell wall components, and the -69 bp still remained half of the increasing tendency of the -314 bp. Thirdly, Using Matlnd and Matlnspector analysis programs, the -575 bp/+14 bp sequence of the hBD-1 gene was searched for potential transcription factor-binding sites in a Transfac transcription factor database (http://tansfac.gbf.de/TRANSFAC) . Multiple high homologous cis elements were found among the sequences from -314 bp to the first exon. They included CCAAT/Enhancer binding protein- ￡ (C/EBP P) in -50 bp region, activator protein-1 (AP-1) in -141 bp region, and CP2 cis element in -197 bp region. Fourthly, in order to determine whether the -69 bp sequences (especially C/EBPp motif) was essential for the induction of hBD-1 gene expression by BCG or not, the gel electrophoresis mobility shift assay (EMSA) was carriedout. Double-stranded oligonucleotide probe containing the C/EBPp binding element was incubated with nuclear extracts from SPC-A-1 cells treated with or without the active BCG cell wall proteins, and electrophoresis mobility shift was analyzed. The result showed that there was a slight difference between the stimulated and unstimulated cells, suggesting that the -69 bp sequences (especially C/EBPP motif) probably took part in the up-regulation of hBD-1 gene by BCG. In summary, BCG cell wall components (18 kDa to 30 kDa) can stimulate hBD-1 gene transcription in pulmonary gland epithelial cells. The sequence (-314 /+14 bp) containing C/EBP P , AP-1, and CP2 motif is responsible for the induction, and a more short sequence, the -69 bp is essential for the enhancement.