Objective: To investigate the involvement of NF-kappaB (NF-kB) regulation of hepcidin gene transcription in acute phase response and its molecular mechanism.
Methods: First, a mouse model of acute phase response was established by intraperitoneal injection of LPS. The relationship between hepcidin expression and dose or time of LPS injection was assessed. Then, electrophoretic mobility shift assay (EMSA) was performed to explore the possibility of the involvement of NF-kB in regulation of hepcidin gene transcription. Next, pAVU6+27-NF-kB, NF-kB p65-specific siRNA expression vector was constructed and transfected into mouse primary hepatocytes using DOTAP liposomal transfection reagents. Hepcidin expression changes after silencing of NF-kB p65 and hepcidin expression after LPS induction were tested.
Results: Hepcidin expression showed a time and dose-dependent manner with regard to LPS injection. At 10 h after 50 microg LPS injection, hepcidin expression reached its peak. The result of EMSA exhibited an evident lag band at -53 - -64 bp, indicating regulation of hepcidin gene expression by NF-kB. After mouse primary hepatocytes were transiently transfected with NF-kB p65-specific siRNAs, Western blot showed that inhibition rate of NF-kB expression was 50%-67%. Hepcidin expression of transfected hepatocytes dropped down obviously in comparison with that of untransfected hepatocytes, and could not be induced by LPS.
Conclusion: Transcription factor NF-kB is likely to be an important molecule in transcription regulation of hepcidin gene. As a key component, p65 subunit binds to hepcidin gene at -53 - -64 bp, and upregulates hepcidin expression.