Autoregulation of Cell-specific MAP Kinase Control of the Tryptophan Hydroxylase Promoter

Abstract

The neurotransmitter serotonin controls a wide range of biological systems, including its own synthesis and release. As the rate-limiting enzyme in serotonin biosynthesis, tryptophan hydroxylase (TPH) is a potential target for this autoregulation. Using the serotonergic neuron-like CA77 cell line, we have demonstrated that treatment with a 5-hydroxytryptamine autoreceptor agonist, CGS 12066A, can lower TPH mRNA levels and promoter activity. We reasoned that this repression might involve inhibition of MAP kinases, since 5-HT1 receptors can increase mitogen-activated protein (MAP) kinase phosphatase levels. To test this hypothesis, we first showed that the TPH promoter can be activated 20-fold by mitogen-activated extracellular-signal regulated kinase kinase kinase (MEKK), an activator of MAP kinases. This activation was then blocked by CGS 12066A. The maximal MAP kinase and CGS repression regulatory region was mapped to between ؊149 and ؊45 base pairs upstream of the transcription start site. The activation by MEKK appears to be cell-specific, because MEKK did not activate the TPH promoter in nonneuronal cell lines. At least part, but not all, of the MAP kinase responsiveness was mapped to an inverted CCAAT box that binds the transcription factor NF-Y. These data suggest a model for the autoregulation of serotonin biosynthesis by repression of MAP kinase stimulation of the TPH promoter. Serotonin (5-hydroxytryptamine; 5-HT)1 is a monoamine neurotransmitter involved in diverse physiological functions including regulation of mood, aggression, anxiety, sleep, satiety, and sexual activity (1). Dysfunction in serotonergic systems has been implicated in the etiology of depression, aggressive behavior, and anxiety disorders (2, 3). The pathophysiological mechanisms behind these illnesses, however, are poorly understood, and very little is known about the control of serotonin levels in neurons.Serotonin biosynthesis is restricted to serotonergic neurons in the brain raphe and gut, the pineal gland, enterochromaffin cells in the gastrointestinal tract, and rodent mast cells (4,5). This is largely due to the cell-specific expression of tryptophan hydroxylase (TPH), the first and rate-limiting step in serotonin biosynthesis, which catalyzes the conversion of tryptophan to 5-hydroxytryptophan. As the rate-limiting enzyme, TPH is a potential target for control of serotonin levels. There is extensive evidence for post-translational regulation of TPH enzyme activity through phosphorylation by Ca 2ϩ /calmodulin-dependent protein kinase, cAMP-dependent protein kinase, and a member of the 14-3-3 protein family (6 -8).Another mechanism for the control of serotonin levels is through a negative feedback loop via activation of presynaptic 5-HT1 autoreceptors, which inhibit neuronal firing and serotonin release (5, 9). Stimulation of 5-HT1 autoreceptors also decreases the conversion of tryptophan to 5-hydroxytryptophan, suggesting that extracellular serotonin can regulate TPH activity (5). No studies to date directly demonstrate that 5...

Activation of the rat dopamine D2 receptor promoter by mitogen‐activated protein kinase and Ca2+/calmodulin‐dependent protein kinase II pathways

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