Takeuchi Yusuke, Miyamoto Eishichi, Fukunaga Kohji
Department of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
J Neurochem. 2002 Oct;83(1):67-79. doi: 10.1046/j.1471-4159.2002.01096.x.
We have reported that the nuclear isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM KII) are involved in the expression of the exon IV-containing brain-derived neurotrophic factor (BDNF) mRNA. We document here the cis-elements and transcription factors responsive to CaM KII in the activation of the promoter upstream of the exon IV (exon IV-BDNF promoter). Effects of constitutively active mutants of CaM KIV, MAPK kinase kinase (MEKK) and protein kinase A (PKA) on the exon IV-BDNF promoter activity were also evaluated by transfection and luciferase assay. The exon IV-BDNF promoter activity was increased by transfection with CaM KII, MEKK and PKA, but not by CaM KIV. Deletion and mutational analysis of the promoter revealed that the region between nucleotides -56 and -27 was responsive to CaM KII, which contained a CCAAT-box in the region between nucleotides -56 and -43. Expression of C/EBPbeta increased the promoter activity and potentiated the effects of CaM KII. The region between nucleotides -79 and -56 was responsive to MEKK, in which both a GA-rich sequence and a GC-box were included. Expression of Sp1 increased the promoter activity, which was further enhanced by transfection with MEKK. The region between nucleotides -43 and -27 was responsive to both PKA and CaM KII, but the transcription factors involved in the region remained unclear. These results suggest that the promoter of the exon IV-BDNF is at least regulated by CaM KII, MEKK and PKA, and that C/EBP/beta and Sp1 are potential transcription factors activated by CaM KII and MEKK, respectively.
我们曾报道，钙/钙调蛋白依赖性蛋白激酶II（CaM KII）的核异构体参与含外显子IV的脑源性神经营养因子（BDNF）mRNA的表达。我们在此记录了外显子IV上游启动子（外显子IV - BDNF启动子）激活过程中对CaM KII有反应的顺式元件和转录因子。还通过转染和荧光素酶测定评估了CaM KIV、丝裂原活化蛋白激酶激酶激酶（MEKK）和蛋白激酶A（PKA）的组成型活性突变体对外显子IV - BDNF启动子活性的影响。用CaM KII、MEKK和PKA转染可增加外显子IV - BDNF启动子活性，但用CaM KIV转染则无此效果。对该启动子的缺失和突变分析表明，核苷酸 - 56至 - 27之间的区域对CaM KII有反应，该区域在核苷酸 - 56至 - 43之间包含一个CCAAT框。C/EBPβ的表达增加了启动子活性并增强了CaM KII的作用。核苷酸 - 79至 - 56之间的区域对MEKK有反应，其中包含一个富含GA的序列和一个GC框。Sp1的表达增加了启动子活性，用MEKK转染可进一步增强该活性。核苷酸 - 43至 - 27之间的区域对PKA和CaM KII均有反应，但该区域涉及的转录因子仍不清楚。这些结果表明，外显子IV - BDNF的启动子至少受CaM KII、MEKK和PKA调控，并且C/EBPβ和Sp1分别是由CaM KII和MEKK激活 的潜在转录因子。
