Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine beta-hydroxylase in SK-N-BE(2)C cells.
Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine beta-hydroxylase in SK-N-BE(2)C cells.
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DOI：
10.1111/j.1471-4159.2009.06260.x
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发表时间：
2009-09
影响因子：
4.7
通讯作者：
中科院分区：
文献类型：
--
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Phox 2a和Phox 2b是两种同源结构域蛋白，在胚胎发育过程中控制去甲肾上腺素能神经元的分化。在本研究中，我们研究了Phox 2a/2b对去甲肾上腺素转运蛋白（NET）和多巴胺β-羟化酶（DBH）（去甲肾上腺素能系统的两个重要标志物）体外表达的可能影响。SK-N-BE（2）C细胞用特异于人Phox 2a和Phox 2b基因的cDNA或短发夹RNA转染。0.1 ~ 5 μg Phox 2a或Phox 2b的cDNA转染显著提高NET和DBH的mRNA和蛋白水平，并呈浓度依赖性。由于转染后NET表达增强，[3 H]去甲肾上腺素的摄取也平行增加。与单独转染Phox 2a或Phox 2b相比，Phox 2a和Phox 2b的共转染没有进一步增加去甲肾上腺素能标记物的表达。Phox 2a或Phox 2b基因特异性shRNA的转染显著降低内源性Phox 2关闭后NET和DBH的mRNA和蛋白水平，这伴随着[3 H]去甲肾上腺素摄取的减少。此外，有一个累加效应后，共转染与特异性的PHox 2a或PHox 2b基因的shRNA NET mRNA水平。最后，Phox 2a特异性shRNA引起的DBH表达减少可以通过转染Phox 2b cDNA来逆转，反之亦然。本研究结果验证了Phox 2a和Phox 2b的NET和DBH在体外的表达和功能的决定性作用。进一步阐明这两种转录因子对去甲肾上腺素能系统关键蛋白的调节作用，可能为衰老引起的去甲肾上腺素能系统功能障碍的治疗开辟新的途径。
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 β-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 μg 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 [3H]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 [3H]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.
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