Low Pyridoxine Concentrations Enhance Lipopolysaccharide-Stimulated Gene Expression of Cyclooxygenase-2 and Inducible Nitric Oxide Synthase in RAW264.7 Cells

Pyridoxal (PL) has been shown to suppress lipopolysaccharide (LPS)-induced gene expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Commonly used cell culture media contain extremely high concentrations of pyridoxine (PN) compared to total serum levels of vitamin B-6. Therefore, we evaluated how physiological concentrations of PN influence LPS-stimulated gene expression of COX-2 and iNOS. The mouse macrophage cell line, RAW264.7, was cultured in PN-free DMEM supplemented with 10% fetal bovine serum (DMEM(-PN+FBS)) for 7 d. Although the level of pyridoxal 5'-phosphate in these cells was decreased by 65%, no change was observed in cell proliferation rate or aspartate aminotransferase activity for 7 d. LPS-induced expression of COX-2 mRNA was compared between DMEM(+FBS) and DMEM(-PN+FBS). COX-2 expression was enhanced by 2.2 or 1.9 times with a 1 or 3 d treatment, respectively; however, no difference was observed at 7 d. PN (0.032-100 mu m) added to the DMEM(-PN+FBS) and RAW264.7 cells was cultured in the medium containing each concentration of PN for 1 d. Enhancement of COX-2 and iNOS gene expression was suppressed by PN addition in a concentration-dependent manner. COX-2 and iNOS mRNA were similarly expressed in cells grown in media containing PN at 4 mu m or higher. Overall, induction of COX-2 and iNOS by LPS was transiently enhanced when RAW264.7 cells were cultured in physiological PN concentrations.