Induction Of GCH1 Expression By LPS

GTP cyclohydrolase, which is encoded by GCH1, catalyses the rate limiting reaction for de novo biosynthesis of 5,6,7,8-tetrahydrobiopterin (BH4). BH4 is an essential coenzyme for aromatic amino acid hydrolase, as well as nitric oxide synthase (NOS), and BH4 deficiency causes decrease in catecholamine synthesis, impaired phenylalanine metabolism and NOS uncoupling, which is closely related with various pathological process, including impaired vascular homeostasis, motor dysfunction. BH4 synthesis can be increased by upregulation of GCH1 expression. Hence, it is highly significant to understand the transcriptional regulatory mechanism of GCH1 gene. GCH1 expresses in multiple tissues and cells and can be induced by various factors. In this study, we used lipopolysaccharide (LPS) to treat mouse macrophage RAW264.7 cells, and GCH activity was stimulated with the expression upregulated. Reporter plasmids with human GCH1 promoter inserted were transfected into RAW264.7 cells, and using the dual luciferase reporter assay, it was revealed that intron 1 is essential for the transcriptional activation of GCH1 promoter by LPS. Furthermore, a 332 bp consensus sequence was found in intron 1 of GCH1 gene among species. Using the reporter assay system, we identified the 332 bp region as an enhancer to be responsible for LPS stimulation. With inhibitors for various well known signaling pathway, it was revealed that MEK1/2 and NF-κB pathway probably were involved in the signaling pathway, rather than PI3K, MAPK, or JNK.Further more, expression of GCH1 in human umbilical vein endothelial (HUVEC) cells was activated by TNFα/IFNγtreatment, in which the 332 bp enhancer was the key element for the response, and NF-κB might be involved in this process as well.In conclusion, in this study we identified a 332 bp enhancer within intron 1 of GCH1 gene, which was responsible for the transcriptional activation by LPS or cytokines, providing new insight for GCH1 transcriptional regulation and treatment for BH4 related diseases.