Abstract
Abstract Human fibroblast growth factor 1 (FGF-1) is involved in the regulation of many biological processes such as tissue growth, wound healing, cell differentiation and angiogenesis. FGF-1B is the major transcript of FGF-1 gene in brain tissue. We previously reported that human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) can be used to monitor endogenous FGF1 expression and neural stem/progenitor cells (NSPCs) isolation. The FGF-1B promoter region contains two regulatory elements (RR1 and RR2), and the minimal region required for the DNA-protein interaction in RR2 is the 18-bp sequence (-484 to -467). Regulatory factor X (RFX) 1 binds to the 18-bp sequence, which has the imperfect palindromic RFX1 consensus sequence, and participates in the transcription regulation in FGF-1B. In the present study, we further showed two other members of the RFX family, RFX2 and RFX3, could bind to the 18-bp sequence. Using electrophoretic mobility shift assay (EMSA), we demonstrated that RFX1 homodimers, RFX1-RFX2, RFX1-RFX3 and RFX2-RFX3 heterodimers bind to the 18-bp sequence. In addition, only the RFX2-RFX3 complex could be detected in the nuclear extract of FGF-1B positive cells, but not in FGF-1B negative cells. Using F1BGFP reporter and siRNA transfection, we found that knockdown of RFX1 or RFX3 could increase F1B promoter activity. In contrast, knockdown of RFX2 with siRNA significantly inhibited F1B promoter activity. Furthermore, by using protein kinase C inhibitors, we demonstrate the involvement of protein kinase C signaling in the regulation of FGF-1B promoter. Detailed mechanisms among PKC signaling, RFX transcription factors and F1B promoter are worthy of further investigation.