Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine beta-hydroxylase in SK-N-BE(2)C cells.
Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 mug of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [(3)H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [(3)H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.
Fan
Yan
Y
Department of Biochemistry and Molecular Biology, School of Medicine, Soochow University Suzhou, China.
Huang
Jingjing
J
Kieran
Niamh
N
Zhu
Meng-Yang
MY
D045744:Cell Line, Tumor; D004299:Dopamine beta-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*
D018398:Homeodomain Proteins; C109220:NBPhox protein; D050484:Norepinephrine Plasma Membrane Transport Proteins; C491061:PHOX2A protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D004299:Dopamine beta-Hydroxylase