Tyrosine hydroxylase gene expression is facilitated by alcohol followed by the degradation of the protein by ubiquitin proteasome system.

OBJECTIVES: Alcohol intake induces brief periods of euphoria; however, its continuous consumption can lead the development of alcohol tolerance. The euphoria, an intense feeling of wellbeing, is deeply associated with dopamine. Dopamine biosynthesis is strictly regulated by tyrosine hydroxylase (TH), a rate-limiting enzyme of dopamine. The aim of this study was to examine the transient or chronic effects of ethanol treatment on TH protein level in vitro. METHODS: Cultured primary mesencephalic neurons were prepared and exposed to 100 mM ethanol for 48 hours or 168 hours. TH and cAMP-responsive element (CRE)-mediated transcriptional activity was measured by reporter gene assay using pTH9.0kb-Luc and pCRE-Luc reporter plasmid. TH protein expression and TH phosphorylation was analyzed by Western blot analysis. Dopamine content was measured by high-performance liquid chromatography (HPLC). RESULTS: Ethanol treatment for 48 hours facilitates TH transcriptional activity and TH protein expression in a cAMP-dependent protein kinase A (PKA) and MAPK/Erk kinase (MEK)-dependent manner in cultured mesencephalic neurons. Ethanol also facilitated TH phosphorylation, which resulted in the elevation of dopamine content. On the other hand, treatment with ethanol for 168 hours did not show significant elevation of TH gene expression and dopamine biosynthesis. Intriguingly, simultaneous treatment with MG-132, a 26S proteasomal inhibitor, recovered the ethanol-induced increase of TH protein expression and dopamine biosynthesis. CONCLUSION: Transient ethanol-treatment facilitates TH gene expression and its phosphorylation in a PKA- and MEK-dependent manner to elevate dopamine biosynthesis, whereas continuous exposure to ethanol abolishes its potent effects on the dopaminergic function to reduce dopamine content. This reduction seems to originate from the decrease of TH protein level by degradation of the protein. Our current data may contribute to the better understanding of alcohol tolerance associated with degradation of TH protein to reduce total-TH level and dopamine biosynthesis.

Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.