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

Abstract

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-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 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.

Profiles of Growth Hormone (GH)-regulated Genes Reveal Time-dependent Responses and Identify a Mechanism for Regulation of Activating Transcription Factor 3 By GH

“…Expression of the genes for GHR (29) and the acid labile subunit, which complexes with circulating insulin-like growth factor-I (30), also increased after 48 h of GH. Expression of early response genes, including Fos, Jun, and Egr1, increased rapidly and transiently within 30 min of GH, as reported previously (31)(32)(33)(34)(35). GH also increased expression of genes encoding negative regulators of GH signaling, including the genes for SOCS-1, SOCS-2, SOCS-3, and cytokine-inducible SH2-containing protein (36 -39).…”

Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade

“…SHC, in turn, bridges EphA2 to GRB2, which facilitates the activation and nuclear translocation of ERK kinases, where they induce the Elk-1 transcription factor kinase, SLAP, SHP2 and FAK (Miao et al, 2000;Pandey et al, 1994Pandey et al, , 1995a, whereas our present study extends signaling from EphA2 at the cell membrane to the nucleus. The induction of Elk-1 is also interesting since ERK stimulation of Elk-1 has been linked with both the postive and negative regulation of cell proliferation and differentiation (Brunet et al, 1995;Clarkson et al, 1999;Davis, 1995;Lin et al, 1997;Macleod et al, 1992;Townsend et al, 1999;Treisman, 1994;Vanhoutte et al, 2001). Although the biological consequences of Elk-1 induction by EphA2 are presently unknown, ligand-mediated activation of EphA2 has been linked with the negative regulation of numerous biological outcomes, including the regulation of cell proliferation, survival, migration, invasion, differentiation and angiogenesis (Miao et al, 2000(Miao et al, , 2001Pandey et al, 1995a,b;Rosenberg et al, 1997;Zantek et al, 1999;Zelinski et al, 2001).…”

Transcriptional Regulation of the Cytosolic Chaperonin θ Subunit Gene, Cctq, by Ets Domain Transcription Factors Elk-1, Sap-1a, and Net in the Absence of Serum Response Factor

“…CQE1 was specifically recognized by the Ets domain transcription factors Elk-1, Sap-1a, and Net, both in vivo and in vitro. Elk-1, Sap-1a, and Net have all been shown to form ternary complexes with SRF on c-fos and/or egr-1 promoters and mediate their transcriptional regulation (27)(28)(29)33). However, binding of ternary complexes to SREs in these immediate early genes is SRF-dependent; thus, TCF subfamily proteins alone exhibit no significant binding to these elements (33).…”

“…The core sequence is essential for Ets factor binding, and its flanking sequences are important for determining the different sequence specificity of these proteins. Elk-1, Sap-1a, or Net forms ternary complexes with serum response factor (SRF) on the serum response elements (SREs) of immediate early gene promoters, including c-fos (27) and egr-1 (28,29), and play important roles in the transcriptional regulation of these genes. These three TCF subfamily proteins share three conserved domains: the DNA-binding domain localized at the amino terminus (30), the SRF-binding domain in the middle portion (31), and the transcriptional activation domain whose activity is stimulated by phosphorylation by MAPK at the carboxyl terminus (32)(33)(34).…”