Rodova Marianna, Nguyen Anh-Nguyet, Blanco Gustavo
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Mol Reprod Dev. 2006 Nov;73(11):1435-47. doi: 10.1002/mrd.20518.
The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.
钠钾ATP酶是质膜中的一种必需酶，在许多依赖于钠离子（Na⁺）和钾离子（K⁺）跨细胞梯度的细胞过程中发挥关键作用。在钠钾ATP酶催化亚基的各种同工型中，α4的表达模式最为有限，仅局限于雄性生殖细胞。α4的活性对于精子功能至关重要，并且在精子发生过程中上调。本研究探讨了人类钠钾ATP酶α4基因（ATP1A4）的转录调控。我们描述了ATP1A4基因的一个5'非翻译区（基于ATP1A4转录起始位点命名为-339/+480）在荧光素酶报告基因检测中具有启动子活性。对该启动子区域的计算机分析揭示了环磷酸腺苷（cAMP）反应元件调节剂（CREM）的共有序列位点（CRE）。因此，二丁酰cAMP（db-cAMP）和CREMtau（CREM的一种睾丸特异性剪接变体）的异位表达能够激活HEK 293 T、JEG-3和GC-1细胞中由ATP1A4启动子驱动的荧光素酶表达。进一步表征db-cAMP和CREMtau对ATP1A4启动子缺失构建体（-339/+80和+25/+480）以及对CRE位点携带突变的-339/+480区域的影响表明，db-cAMP和CREMtau的作用需要位于转录起始位点上游263 bp处的CRE基序。电泳迁移率变动分析（EMSA）实验证实该CRE序列是一个真正的CREMtau结合位点。这些结果首次证明了cAMP和CREMtau对ATP1A4基因表达的转录调控，CREMtau是雄性生殖细胞基因表达所必需的转录因子。
