In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis. Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (-624 to -250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-l and Sap-la. Coexpression of Sap-la and Elk-l resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.
机构:
Univ Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Shandong Univ, Inst Cell Biol, Sch Med, Jinan 250021, Peoples R ChinaUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Wang, Xiangdong
Yang, Ning
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Yang, Ning
Deng, Luqin
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Cell Biol, Birmingham, AL 35294 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Deng, Luqin
Li, Xin
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Pathol, Birmingham, AL 35294 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Li, Xin
Jiang, Jing
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Jiang, Jing
Gan, Yujun
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Cell Biol, Birmingham, AL 35294 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Gan, Yujun
Frank, Stuart J.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA
Univ Alabama, Dept Cell Biol, Birmingham, AL 35294 USA
Vet Adm Med Ctr, Endocrinol Sect, Med Serv, Birmingham, AL 35233 USAUniv Alabama, Dept Med, Div Endocrinol Diabet & Metab, Birmingham, AL 35294 USA