He Zuping, Jiang Jiji, Kokkinaki Maria, Golestaneh Nady, Hofmann Marie-Claude, Dym Martin
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20057, USA.
Stem Cells. 2008 Jan;26(1):266-78. doi: 10.1634/stemcells.2007-0436. Epub 2007 Oct 25.
Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in regulating the proliferation of spermatogonial stem cells (SSC). The signaling pathways mediating the function of GDNF in SSC remain unclear. This study was designed to determine whether GDNF signals via the Ras/ERK1/2 pathway in the C18-4 cells, a mouse SSC line. The identity of this cell line was confirmed by the expression of various markers for germ cells, proliferating spermatogonia, and SSC, including GCNA1, Vasa, Dazl, PCNA, Oct-4, GFRalpha1, Ret, and Plzf. Western blot analysis revealed that GDNF activated Ret tyrosine phosphorylation. All 3 isoforms of Shc were phosphorylated upon GDNF stimulation, and GDNF induced the binding of the phosphorylated Ret to Shc and Grb2 as indicated by immunoprecipitation and Western blotting. The active Ras was induced by GDNF, which further activated ERK1/2 phosphorylation. GDNF stimulated the phosphorylation of CREB-1, ATF-1, and CREM-1, and c-fos transcription. Notably, the increase in ERK1/2 phosphorylation, c-fos transcription, bromodeoxyuridine incorporation, and metaphase counts induced by GDNF, was completely blocked by pretreatment with PD98059, a specific inhibitor for MEK1, the upstream regulator of ERK1/2. GDNF stimulation eventually upregulated cyclin A and CDK2 expression. Together, these data suggest that GDNF induces CREB/ATF-1 family member phosphorylation and c-fos transcription via the Ras/ERK1/2 pathway to promote the proliferation of SSC. Unveiling GDNF signaling cascades in SSC has important implications in providing attractive targets for male contraception as well as for the regulation of stem cell renewal vs. differentiation.
胶质细胞系源性神经营养因子（GDNF）在调节精原干细胞（SSC）增殖中起关键作用。介导GDNF在SSC中功能的信号通路仍不清楚。本研究旨在确定GDNF是否通过Ras/ERK1/2信号通路在C18-4细胞（一种小鼠SSC系）中发挥作用。通过多种生殖细胞、增殖期精原细胞和SSC标志物的表达，包括GCNA1、Vasa、Dazl、PCNA、Oct-4、GFRalpha1、Ret和Plzf，证实了该细胞系的特性。蛋白质免疫印迹分析显示，GDNF激活了Ret酪氨酸磷酸化。GDNF刺激后，Shc的所有3种异构体均被磷酸化，免疫沉淀和蛋白质免疫印迹表明，GDNF诱导磷酸化的Ret与Shc和Grb2结合。GDNF诱导了活性Ras的产生，进而激活了ERK1/2磷酸化。GDNF刺激了CREB-1、ATF-1和CREM-1的磷酸化以及c-fos转录。值得注意的是，GDNF诱导的ERK1/2磷酸化增加、c-fos转录、溴脱氧尿苷掺入和中期细胞计数，被MEK1（ERK1/2的上游调节因子）的特异性抑制剂PD98059预处理完全阻断。GDNF刺激最终上调了细胞周期蛋白A和CDK2的表达。总之，这些数据表明，GDNF通过Ras/ERK1/2信号通路诱导CREB/ATF-1家族成员磷酸化和c-fos转录，以促进SSC的增殖。揭示SSC中的GDNF信号级联对于提供男性避孕以及干细胞更新与分化调节的有吸引力靶点具有重要意义。
