cAMP-Response Element (CRE)-Mediated Transcription by CRE-Binding Protein (CREB) Is Essential for Human Tryptophan...

Human tryptophan hydroxylase 2 (hTPH2), a rate-limiting enzyme in the synthesis of central serotonin, is dysregulated in various neuropsychiatric disorders. However, the regulatory mechanisms underlying TPH2 gene expression remain unknown. Here, we aimed to analyze the cAMP signaling-mediated activation of hTPH2 promoter activity involving an inverted cAMP-response element (CRE; 5'-TAACGTCA-3'; -243/-236 relative to the transcription start site, +1). A 2-kb region of the TPH2 gene (-1850/+141; except +10/+121, a region containing repression elements) was cloned into pGL4-Basic to construct a luciferase reporter plasmid. Promoter activity was assessed via transient transfection of RN46A cells derived from rat raphe neurons. Forskolin-induced increase in cAMP levels enhanced hTPH2 promoter activity, which was significantly suppressed by the protein kinase A (PKA) inhibitor, H-89, or CRE mutants. Gel mobility shift assays and chromatin immunoprecipitation assays confirmed the specific binding of CRE-binding protein (CREB) to CRE. hTPH2 promoter activity was decreased by endogenous CREB knockdown. Overexpression of PKAα catalytic subunit and CREB tended to increase the hTPH2 promoter activity. Furthermore, overexpression of CREB-regulated transcription co-activator 1 (CRTC1) the principal CRTC isoform in the brain, with PKAα/CREB significantly increased the hTPH2 promoter activity. In contrast, under PKAα/CRTC1 overexpression conditions, hTPH2 promoter activity was slightly decreased by the overexpression of R133A-CREB (defective for phosphorylation by PKA) and further decreased by the overexpression of R314A-CREB (defective for interaction with CRTC). Overall, our results suggest that high cAMP levels regulate the hTPH2 promoter activity mainly via PKA and its downstream effectors, such as CREB. Moreover, PKA-CRTC signaling stimulates TPH2 gene transcription upon CREB activation.