Increase Of β-defensin-3 Gene Expression In Mouse Liver And NF-κB Regulation During The Acute-phase Response To Lipopolysaccharide

Antibacterial peptides have been recognized as important mediators of innate immunity against microbes. To date , a large variety of antibacterial peptides are expressed in a series of tissues and cells of mammals and some mature peptides with antibacterial activity have been isolated. In addition to their role in immunity, some antibacterial peptides can play the distinct roles in cell differentiation, tissue repair and antitumor immunity. Acute phase respo- nse mediators can act on the hepatocytes by non-specific stimulation, which may lead to the changes of the gene expression and protein synthesis. Some antibacterial peptides such as PR-39, a member of Cathelicidin family, can also be induced and expressed in liver as an acute-phase reactant. Mouse β -defensin -3 (mBD3) is only constitutively expressed in mucosal tissues. In order to study the expression of 3 -defensins in liver as acute phase response proteins, a murine systemic acute phase responsive model was established by intraperitoneal injection of lipopolysaccharide(LPS) in our study. The mBD3 cDNA sequence (145-169 bp) was labled with [ -32P]ATP as a probe to detect mBD3 mRNA in different tissues by Northern blot and analyze the time- and dose- dependant expression caused by LPS.The 5' flanking sequence(-167-179 bp) of the mBD3 gene was designed as the probe and labled with [ -32P] dCTP to investigate the binding of transcriptional factors to this region by electrophoretic mobility shift assay (EMSA) and South-western blot. Results :1. During a systemic acute-phase response resulted by intraperitonealinjection of LPS the expression of mBD3 gene was only detected in mouse liver and not in heart, lung, spleen and kidney by Northern blot.2. The minimum dose of LPS inducing mBD3 expression was 1.5βg/g, although the mBD3 mRNA level tended to be dose-dependant, there were no distinct difference between the relative pixel values of the autoradiographical signal.3. The mBD3 mRNA was detected every 2 hours after LPS injection at the dose of 1.5βg/g. It was found that mBD3 gene was not expressed until 6 hours later and the mRNA level reached maximum at 8 or 10 hours, then decreased 12 hours later.4. The results of EMSA showed the obvious retardant bands, which suggest that some proteins in nuclei can bind to the specific DNA sequence of mBD3 gene after LPS treatment.5. The molecular mass of this DNA binding protein was assessed between 43.0-66.2 KD by South-Western blot.6. EMSA with coincubation of specific antibodies to the p50 or p65 subunits of NF- к B showed a distinct retardation in the mobility with the p65 antibody and a reduction in the intensity of the shifted band with the p50 antibody. These results suggest that the transcriptional factor is the p50-p65 heterodimer of NF- к B.Conclusion:1. The expression of mBD3 gene can be induced in liver during the systemic acute-phase response to the challenge by LPS; 2. The acute phase-related mBD3 gene expression appears to be time- and dose-dependant of LPS stimulation; 3. The mechanism of induction likely involves the binding of NF- к B to the regulation sequence of the mBD3 gene.