Ras/MEK pathway is required for NGF-induced expression of tyrosine hydroxylase gene

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Abstract

Neurotrophins are essential for the development and survival of catecholaminergic neurons. However, the critical pathway for expression of the tyrosine hydroxylase (TH) gene induced by neurotrophin is still unclear. Here we found that Ras/MEK pathway is required for NGF-induced expression of the TH gene in PC12D cells. Induction of TH mRNA by NGF was abolished by pretreatment of the cells with U0126, an inhibitor for MEK1/2, but not with inhibitors for p38 MAPK, PI3K, and PKA. U0126 inhibited TH promoter activity at the same concentration as it acted on ERK1/2 phosphorylation. A dominant-negative form of Ras suppressed the NGF-induced activation of the TH reporter gene, and transient transfection of cells with wild-type Ras and an active form of MEK1 increased the TH promoter activity. The reporter assay also demonstrated that the Ras/MEK pathway acted on both the AP-1-binding motif and the cAMP-responsive element in the TH promoter.

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Materials and methods

Plasmids. Firefly luciferase reporter genes containing mouse TH promoter regions (mTHpro4.3-Luc, mTHpro0.8-Luc, and mTHpro0.8CREmt-Luc) were constructed as previously described [24]. A mutation in the AP-1-binding motif of mTHpro0.8-Luc (GAATTCA) was generated by a PCR method using a forward primer (TAGCCCGGGCTCGAGACCATGATGCAGG) and a mutated reverse primer (TTAGATCTAATTGCATCCACTGTCGCAGGCACCTGCCTCTGAATTCCCCTCCGCCCTAG ACACG) and the wild-type vector, mTHpro4.3-Luc (TGATTCA). The mutation was

An inhibitor of MEK blocked induction of TH mRNA in response to NGF

First, we examined induction of TH mRNA by NGF in PC12D cells. Real-time PCR analysis demonstrated that induction of TH mRNA was detectable within 1 h after exposure to NGF. The level reached its maximum at 2–3 h and remained there for at least 24 h after the start of exposure (Fig. 1A). To explore the signaling cascades activated by NGF to regulate the TH gene expression, we examined the effect of inhibitors specific for protein kinases known to be activated in response to NGF. We found that the

Discussion

In the present study, we demonstrated that the Ras/MEK pathway was required for the NGF-mediated transcriptional activation to express the TH gene in PC12D, a subclone of the PC12 cell line. We also showed that the Ras/MEK pathway acted on AP1 and CRE in the TH promoter to transcribe the TH gene in response to NGF.

NGF has been demonstrated to up-regulate the TH gene expression in peripheral sympathetic and sensory neurons. In addition, TrkB ligands, i.e., BDNF and NT-4/5, recently have been

Acknowledgements

This work was supported by grants from the programs Grants-in-Aid for Encouragement of Young Scientists (to T.S.) and Grants-in-Aid for Scientific Research on Priority Areas (C)—Advanced Brain Science Project—(to H.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Health Science Research Grants—Research on Human Genome, Tissue Engineering, Food Biotechnology—from the Ministry of Health, Labour and Welfare of Japan (to H.I.); and by the Human Frontier Science

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Abbreviations: TH, tyrosine hydroxylase; NGF, nerve growth factor; MAPK, mitogen activated protein kinase; ERK, extracellular signal regulated kinase; MEK, MAP and ERK kinase; PI3K, phosphatidylinositol 3-kinase; CRE, cAMP-responsive element; CREB, CRE-binding protein; AP1, AP-1-binding site; PKA, protein kinase A; kb, kilobase(s); bp, base pair(s); FSK, forskolin.

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