Ono T, Inokuchi K, Ogura A, Ikawa Y, Kudo Y, Kawashima S
Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
J Biol Chem. 1997 May 30;272(22):14404-11. doi: 10.1074/jbc.272.22.14404.
To identify genes whose expression is neuronal activity-dependent, we used an mRNA differential display technique and discovered that parathyroid hormone-related protein (PTHrP) is expressed in an activity-dependent manner in primary cultures of rat cerebellar granule neurons. PTHrP mRNA was expressed as early as 1 h by the addition of KCl to a final concentration of 25 mM to the culture medium. This expression was induced by Ca2+ influx through voltage-dependent L-type Ca2+ channels and regulated at the transcriptional step. PTHrP mRNA was persistently expressed before and after the time of commitment of granule neurons to apoptosis when they are cultured in the presence of 25 mM KCl or both 150 microM N-methyl-D-aspartic acid and 15 mM KCl, both of which promote the survival of these neurons. PTHrP was rapidly secreted into the culture medium in a depolarization-dependent manner. Parathyroid hormone/PTHrP receptor mRNA was also expressed in the primary cultures, and its expression was up-regulated by KCl and/or N-methyl-D-aspartic acid. The addition of anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. These results suggest that PTHrP is involved in an autocrine loop and required for the survival of granule neurons.
为了识别其表达依赖于神经元活动的基因，我们采用了mRNA差异显示技术，并发现甲状旁腺激素相关蛋白（PTHrP）在大鼠小脑颗粒神经元的原代培养物中以活动依赖的方式表达。通过向培养基中添加KCl至最终浓度25 mM，PTHrP mRNA早在1小时就开始表达。这种表达是由通过电压依赖性L型Ca2+通道的Ca2+内流诱导的，并在转录水平受到调控。当颗粒神经元在25 mM KCl或150 microM N-甲基-D-天冬氨酸和15 mM KCl存在下培养时，PTHrP mRNA在颗粒神经元发生凋亡之前和之后持续表达，这两种情况都能促进这些神经元的存活。PTHrP以去极化依赖的方式迅速分泌到培养基中。甲状旁腺激素/PTHrP受体mRNA也在原代培养物中表达，其表达受到KCl和/或N-甲基-D-天冬氨酸的上调。向培养基中添加抗PTHrP抗血清导致颗粒神经元的活动依赖性存活减少。这些结果表明，PTHrP参与自分泌环路，是颗粒神经元存活所必需的。
