Neuron-specific calcium-dependent induction of tryptophan hydroxylase-2 (TPH2) transcription

F. REMES LENICOV; P. R. ALBERT

Banff, Canada

Conferencia; 16th Biennial Meeting of the International Society for Neural Development; 2006

International Society for Neural Development

Serotonin (5-HT) modulates developmental events, such as cell division, differentiation and migration. Disruption of 5-HT neurotransmission during development is known to have long-term effects leading to altered behavior. Neuronal tryptophan hydroxylase (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis in the brain, therefore transcriptional control of TPH2 expression is expected to influence the 5-HT output of serotonergic neurons. Due to its recent discovery, the transcriptional regulation of this key enzyme has not been characterized. To study TPH2 gene expression we have used primary cultures from rat embryonic brainstem and RN46A cells, a cell line derived from embryonic rat raphe neurons that can be differentiated to express an enhanced serotonergic phenotype. We quantified endogenous TPH2 RNA levels by real time RT-PCR. Depolarization of neurons in primary cultures using 40mM KCl increased TPH2 RNA levels 1.7 times within 1 h. Equal treatment of the RN46A cells induced a 15-fold increase. Similarly, Ca++ mobilization using ionomycin increased TPH2 transcription up to 4-fold within 3 h, suggesting a key role for calcium in the induction of TPH2 RNA. In order to find DNA elements responsible of this effect, we identified the TPH2 promoter and transfected it into RN46A cells. In this model, ionomycin induced a 1.6-fold increase in relative luciferase activity. We narrowed the location of the responsive element to the first 280 base pairs upstream of the start site of transcription. Interestingly, Ca++ mobilization did not induce transcription in a TPH2-negative cell line, or in a non-neuronal, TPH2-positive cell line.  We conclude that in neuronal cell types, TPH2 expression is induced by calcium mobilizing agents. This effect is mediated by elements located in the proximal TPH2 promoter region. Activity-dependence of 5-HT production might be an important characteristic for the role of the serotonergic system in development.