Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced transcription of egr-1 (early growth response factor-1) in 3T3-F442A preadipocytes

In our search for transcription factors induced by GH, we have analyzed imm ediate early gene activation in a model of GH-dependent differentiation. He re we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors respons ible 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 appea red not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA syn thesis. 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-F4 42A cells and approximately 90% inducibility in CHO-K1 cells. This region c ontains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-s pecific mutations of individual SRE and Ets sites within this region each r educed GH inducibility of the promoter. Use of these site-specific mutation s in EMSA showed that disruption of either Ets or SRE sites abrogated terna ry complex formation at the composite sites. DNA binding of ternary complex es, 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 pr omoter activity, although transfection with expression vectors for either E ts factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of t hese Ets factors at multiple sites on the promoter and that stabilization o f ternary complexes with SRF at these sites maximizes this response.