Badinga L, Song S, Simmen R C, Simmen F A
Cell Biology Center, Biotechnology Institute, Department of Animal Science, University of Florida, Gainesville 32611-0920, USA.
Endocrine. 1998 Jun;8(3):281-9. doi: 10.1385/ENDO:8:3:281.
Insulin-like growth factor binding protein-2 (IGFBP-2), the predominant IGFBP in the fetal circulation and an induced protein during several types of malignancies, belongs to a family of structurally related proteins that bind the mitogens, IGF-1 and IGF-2. The present study focused on functional analysis of the 5 '-flanking region (approximately 1.3 kb) of the IGFBP-2 gene to identify nuclear factors that mediate hepatic transcription of this gene. Luciferase (LUC) reporter constructs containing progressive deletions of 5'-flanking DNA and the intact promoter of the porcine IGFBP-2 gene were examined for functional activity by transient transfection of human HepG2 liver cells. LUC activity of the transfected reporter gene driven by the IGFBP-2 promoter and flanking sequences to -1397 (numbering relative to initiation codon at +1) was 22-fold higher than that of promoterless parent LUC vector. This activity was decreased by 60% with deletion of sequences to -874 bp, and dropped to basal levels with further truncation to -764 bp. The region between -874 and -765 bp (110 bp) functioned as a potent stimulator of heterologous SV40 promoter activity (110 bp/SV40-LUC construct) and was found to contain two noncontiguous basic helix-loop-helix (bHLH) transcription factor binding motifs (E-boxes [CAN NTG]: CACCTG and CAAATG). In electrophoretic mobility shift assays, nuclear proteins prepared from HepG2 cells formed two complexes (C1, C2) with double-stranded oligonucleotides containing either HLH sequence, mutations of which resulted in loss of complex formation. Southwestern blot analysis identified an HepG2 nuclear protein with molecular mass of 48 kDa, similar to that of the bHLH transcription factor AP-4, which bound the CACCTG motif. Cotransfection of HepG2 cells with the 110-bp/SV40-LUC construct and an expression vector encoding human AP-4 increased IGFBP-2 fragment-dependent SV40 promoter activity by 16-fold. This AP-4-mediated stimulation was lost following block mutation of both bHLH motifs within the IGFBP-2 110-bp fragment. Results demonstrate the functional importance of sequences upstream of the promoter in IGFBP-2 gene transcription and identify a novel mechanism by which bHLH proteins potentially may affect cell proliferation and differentiation via induction of IGFBP-2 synthesis.
胰岛素样生长因子结合蛋白-2（IGFBP-2）是胎儿循环中主要的IGFBP，也是几种恶性肿瘤中的诱导蛋白，属于与有丝分裂原IGF-1和IGF-2结合的结构相关蛋白家族。本研究聚焦于IGFBP-2基因5'侧翼区（约1.3 kb）的功能分析，以鉴定介导该基因肝脏转录的核因子。通过瞬时转染人HepG2肝细胞，检测含有猪IGFBP-2基因5'侧翼DNA逐步缺失片段和完整启动子的荧光素酶（LUC）报告基因构建体的功能活性。由IGFBP-2启动子和侧翼序列驱动至-1397（相对于起始密码子+1编号）的转染报告基因的LUC活性比无启动子的亲本LUC载体高22倍。缺失至-874 bp序列时，该活性降低60%，进一步截短至-764 bp时降至基础水平。-874至-765 bp之间的区域（110 bp）作为异源SV40启动子活性的有效刺激物（110 bp/SV40-LUC构建体），并发现含有两个不连续的碱性螺旋-环-螺旋（bHLH）转录因子结合基序（E盒[CAN NTG]：CACCTG和CAAATG）。在电泳迁移率变动分析中，从HepG2细胞制备的核蛋白与含有任一HLH序列的双链寡核苷酸形成两种复合物（C1、C2），其突变导致复合物形成丧失。蛋白质印迹分析鉴定出一种分子量为48 kDa的HepG2核蛋白，类似于bHLH转录因子AP-4，它与CACCTG基序结合。将HepG2细胞与110-bp/SV40-LUC构建体和编码人AP-4的表达载体共转染，可使IGFBP-2片段依赖性SV40启动子活性提高16倍。在IGFBP-2 110-bp片段内的两个bHLH基序发生阻断突变后，这种AP-4介导的刺激作用丧失。结果证明了启动子上游序列在IGFBP-2基因转录中的功能重要性，并确定了一种新机制，即bHLH蛋白可能通过诱导IGFBP-2合成来影响细胞增殖和分化。
