Tetrahydrobiopterin biosynthesis in C6 glioma cells: Induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment

The possiblity that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 mu g/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.