Karki Pratap, Johnson James, Son Deok-Soo, Aschner Michael, Lee Eunsook
Department of Physiology, School of Medicine, Meharry Medical College, Nashville, TN, 37208, USA.
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
Mol Neurobiol. 2017 Mar;54(2):964-976. doi: 10.1007/s12035-016-9705-9. Epub 2016 Jan 21.
Transforming growth factor-alpha (TGF-α) is known to play multifunctional roles in the central nervous system (CNS), including the provision of neurotropic properties that protect neurons against various neurotoxic insults. Previously, we reported that TGF-α mediates estrogen-induced enhancement of glutamate transporter GLT-1 function in astrocytes. However, the regulatory mechanism of TGF-α at the transcriptional level remains to be established. Our findings revealed that the human TGF-α promoter contains consensus sites for several transcription factors, such as NF-κB and yin yang 1 (YY1). NF-κB served as a positive regulator of TGF-α promoter activity, corroborated by observations that overexpression of NF-κB p65 increased, while mutation in the NF-κB binding sites in the TGF-α promoter reduced the promoter activity in rat primary astrocytes. Pharmacological inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC; 50 μM) or quinazoline (QNZ; 10 μM) also abolished TGF-α promoter activity, and NF-κB directly bound to its consensus site in the TGF-α promoter as evidenced by electrophoretic mobility shift assay (EMSA). Dexamethasone (DX) increased TGF-α promoter activity by activation of NF-κB. Treatment of astrocytes with 100 nM of DX for 24 h activated its glucocorticoid receptor and signaling proteins, including MAPK, PI3K/Akt, and PKA, via non-genomic pathways, to enhance TGF-α promoter activity and expression. YY1 served as a critical negative regulator of the TGF-α promoter as overexpression of YY1 decreased, while mutation of YY1 binding site in the promoter increased TGF-α promoter activity. Treatment for 3 h with 250 μM of manganese (Mn), an environmental neurotoxin, decreased astrocytic TGF-α expression by activation of YY1. Taken together, our results suggest that NF-κB is a critical positive regulator, whereas YY1 is a negative regulator of the TGF-α promoter. These findings identify potential molecular targets for neurotherapeutics that may modulate TGF-α regulation and afford neuroprotection.
已知转化生长因子α（TGF-α）在中枢神经系统（CNS）中发挥多种功能作用，包括提供神经营养特性以保护神经元免受各种神经毒性损伤。此前，我们报道TGF-α介导雌激素诱导的星形胶质细胞中谷氨酸转运体GLT-1功能增强。然而，TGF-α在转录水平的调控机制仍有待确定。我们的研究结果表明，人TGF-α启动子含有几个转录因子的共有位点，如核因子κB（NF-κB）和阴阳1（YY1）。NF-κB作为TGF-α启动子活性的正调控因子，这一点得到了以下观察结果的证实：NF-κB p65的过表达增加了启动子活性，而TGF-α启动子中NF-κB结合位点的突变则降低了大鼠原代星形胶质细胞中的启动子活性。用吡咯烷二硫代氨基甲酸盐（PDTC；50 μM）或喹唑啉（QNZ；10 μM）对NF-κB进行药理抑制也消除了TGF-α启动子活性，并且电泳迁移率变动分析（EMSA）证明NF-κB直接与其在TGF-α启动子中的共有位点结合。地塞米松（DX）通过激活NF-κB增加了TGF-α启动子活性。用100 nM的DX处理星形胶质细胞24小时，通过非基因组途径激活其糖皮质激素受体和信号蛋白，包括丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）和蛋白激酶A（PKA），以增强TGF-α启动子活性和表达。YY1作为TGF-α启动子的关键负调控因子，因为YY1的过表达降低了启动子活性，而启动子中YY1结合位点的突变则增加了TGF-α启动子活性。用250 μM的锰（Mn），一种环境神经毒素，处理3小时，通过激活YY1降低了星形胶质细胞中TGF-α的表达。综上所述，我们的结果表明NF-κB是关键的正调控因子，而YY1是TGF-α启动子的负调控因子。这些发现确定了神经治疗学的潜在分子靶点，这些靶点可能调节TGF-α的调控并提供神经保护。
