National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter Medical Center, Bronx, NY, United States.
Biochem Biophys Res Commun. 2013 Aug 2;437(3):355-60. doi: 10.1016/j.bbrc.2013.06.079. Epub 2013 Jun 28.
The ankryn repeat domain proteins, Ankrd1 and Ankrd2, are expressed at the highest levels in skeletal muscle and heart where they are localized to the I band of the sarcomere through binding to titin and myopaladin. Ankrd1 and Ankrd2 migrate from the sarcomere to the nucleus when muscle is stressed, and act as coregulators for a growing number of transcription factors. Expression of Ankrd1 is altered by castration suggesting a link to androgen action. This investigation explored the effects of testosterone on Ankrd1 and Ankrd2 expression and determined whether Ankrd1 or Ankrd2 binds to or regulates the transcriptional activity of the androgen receptor (AR). Incubation of rat L6 myoblasts expressing the human AR (L6.AR) with testosterone reduced mRNA levels for Ankrd1 by approximately 50% and increased those for Ankrd2 by 20-fold. In reporter gene assays conducted with CHO cells co-transfected with an ARE-Luc reporter gene, Ankrd1 blocked the ability of testosterone to increase reporter gene activity while Ankrd2 had no effect. The effect of Ankrd1 and Ankrd2 on repression of the MAFbx promoter by testosterone was also tested in C2C12 cells using an MAFbx-Luc reporter gene (pMAF400-Luc); Ankrd1 blocked repression of pMAF400-Luc by testosterone while Ankrd2 did not. Co-immunoprecipitation studies revealed that Ankrd1 bound to the AR whereas Ankrd2 did not. The effect of Ankrd1 or Ankrd2 on changes in gene expression induced by testosterone in L6.AR cells was also evaluated. Incubation of L6.AR cells with testosterone modestly reduced myogenin mRNA levels but did not significantly alter those for mdm2, MEF2d, TnnI1, TnnI2, or p21. When cells were transfected with Ankrd1, testosterone markedly reduced mRNA levels for MEF2d, myogenin, p21 and TnnI1, increased those for TnnI2, but did not alter those for mdm2. When cells were transfected with Ankrd2, testosterone increased MEF2d and myogenin mRNA levels, having the opposite effect to cells transfected with Ankrd1; Ankrd2 did not change the effects of testosterone on TnnI1, TnnI2, p21, or mdm2 mRNA levels. In conclusion, testosterone regulates the expression of Ankrd1 and Ankrd2; Ankrd1 binds to and directly regulates the transcriptional activity of the AR whereas Ankrd2 does not; expression levels of both Ankrd1 and Ankrd2 modulate effects of testosterone on gene expression in cultured myoblasts.
ankryn 重复结构域蛋白 ankrd1 和 ankrd2 在骨骼肌和心脏中表达水平最高，通过与肌联蛋白和肌钙蛋白 paladin 结合，定位于肌节的 I 带。当肌肉受到压力时，ankrd1 和 ankrd2 从肌节迁移到细胞核，并作为越来越多转录因子的共调节剂。ankrd1 的表达受阉割的影响，提示其与雄激素作用有关。本研究探讨了睾酮对 ankrd1 和 ankrd2 表达的影响，并确定 ankrd1 或 ankrd2 是否与雄激素受体 (ar) 的转录活性结合或调节。用睾酮孵育表达人 ar 的大鼠 l6 成肌细胞 (l6.ar)，可使 ankrd1 的 mrna 水平降低约 50%，ankrd2 的 mrna 水平增加 20 倍。在与雄激素反应元件-luc 报告基因共转染的 cho 细胞中进行的报告基因实验中，ankrd1 阻断了睾酮增加报告基因活性的能力，而 ankrd2 没有影响。在使用 mafbx-luc 报告基因 (pmaf400-luc) 的 c2c12 细胞中，还测试了 ankrd1 和 ankrd2 对 mafbx 启动子被睾酮抑制的影响；ankrd1 阻断了睾酮对 pmaf400-luc 的抑制作用，而 ankrd2 没有。免疫共沉淀研究表明 ankrd1 与 ar 结合，而 ankrd2 不与 ar 结合。还评估了 ankrd1 或 ankrd2 对 l6.ar 细胞中睾酮诱导的基因表达变化的影响。用睾酮孵育 l6.ar 细胞可使肌球蛋白 mrna 水平适度降低，但对 mdm2、mef2d、tnn11、tnn12 或 p21 的水平没有显著影响。当细胞转染 ankrd1 时，睾酮明显降低 mef2d、myogenin、p21 和 tnn11 的 mrna 水平，增加 tnn12 的 mrna 水平，但不改变 mdm2 的 mrna 水平。当细胞转染 ankrd2 时，睾酮增加 mef2d 和肌球蛋白 mrna 水平，与转染 ankrd1 的细胞相反；ankrd2 不改变睾酮对 tnn11、tnn12、p21 或 mdm2 mrna 水平的影响。总之，睾酮调节 ankrd1 和 ankrd2 的表达；ankrd1 与 ar 结合并直接调节其转录活性，而 ankrd2 则不调节；ankrd1 和 ankrd2 的表达水平调节睾酮对培养的成肌细胞中基因表达的影响。
