Nerve growth factor down-regulates the expression of norepinephrine transporter in rat pheochromocytoma (PC12) cells.
Functional expression of norepinephrine transporter (NET) and its regulation were examined in rat pheochromocytoma cell line, PC12. Nerve growth factor (NGF) decreased [3H]-norepinephrine (NE) uptake in association with a decrease in NET mRNA levels. On the other hand, levels of tyrosine hydroxylase mRNA increased in PC12 cells treated with NGF for 4-24 h, while Oct-2 mRNA levels decreased at 4 h with NGF then recovered for 8-24 h in the presence of NGF. Both bFGF and EGF reduced [3H]NE uptake, although they failed to affect NET mRNA levels. To examine the NET transcriptional regulation, we identified the 5'-noncoding region of rat NET mRNA by the rapid amplification of cDNA end (RACE) method and cloned the 5'-flanking region of NET gene. The newly identified exon encodes the untranslated region of rat NET mRNA upstream of the known 5'-region including ATG start codon. Constructs having green fluorescent protein (GFP) as reporter were made with the cloned NET gene, and promoter activity was examined in CHO and SK-N-SH cells transiently transfected and in PC12 cells stably transfected with NET-GFP constructs. The results indicate that the 2.1 kb NET flanking region displays promoter activity and is responsible for the NGF-induced down-regulation of NET expression.
Ikeda
T
T
Department of Pharmacology, Hiroshima University Faculty of Dentistry, Kasumi 1-2-3, Minamiku, 734-8553, Hiroshima, Japan.
Kitayama
S
S
Morita
K
K
Dohi
T
T
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009474:Neurons / Q000502:physiology*; D009638:Norepinephrine / Q000493:pharmacokinetics; D050484:Norepinephrine Plasma Membrane Transport Proteins; D050813:Octamer Transcription Factor-2; D016716:PC12 Cells; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D013566:Sympathomimetics / Q000493:pharmacokinetics; D027981:Symporters; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology; D014316:Tritium; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D020121:5' Untranslated Regions; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D050813:Octamer Transcription Factor-2; C489205:Pou2f2 protein, rat; D012333:RNA, Messenger; C493226:Slc6a2 protein, rat; D013566:Sympathomimetics; D027981:Symporters; D014157:Transcription Factors; D014316:Tritium; D020932:Nerve Growth Factor; D014446:Tyrosine 3-Monooxygenase; D009638:Norepinephrine