Egr1 Luciferase Reporter Lentivirus

Binding of insulin to the insulin receptor triggers intracellular signaling cascades, including activation of protein and lipid kinases. As a result, multiple biological functions of the cell are altered. Here, the researchers analyzed the regulatory and signaling cascades leading to insulin-induced activation of stimulus-responsive transcription factors. The results show that stimulation of the insulin receptor induces the expression of the transcription factor Egr-1. Upon expression of a dominant negative mutant of the ternary complex factor Elk-1, an attenuation of Egr-1 promoter activation was observed. These data were confirmed by experiments showing that insulin receptor stimulation increases the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the Elk-1 dominant negative mutant reduced insulin-induced AP-1 activation, indicating that Elk-1 controls both serum response element- and AP-1-regulated transcription. In addition, stimulation of the insulin receptor activates transcription controlled by the cyclic AMP response element (CRE), which involves the transcription factor CREB.

Here, the researchers analyzed the Egr-1 promoter-controlled luciferase reporter genes Egr-1.2.luc and Egr-1.1luc (Figure 1B) to identify genetic elements within the Egr-1 gene that function as insulin response elements. They inserted the Egr-1 promoter/luciferase reporter into the chromatin of HIRcB cells by lentiviral gene transfer. Stimulation of HIRcB cells with insulin induced transcription of the Egr-1.2.luc reporter gene by approximately 9.7-fold (Figure 1C, left). Stimulation of cells with insulin also induced transcription of the Egr-1.1.luc reporter gene, but to a much lesser extent (3.2-fold, Figure 1C, right). These results suggest that the number of SREs is important for insulin stimulation of the Egr-1 promoter. The researchers directly measured the insulin responsiveness of SREs by analyzing the Egr-1SRE.luc reporter gene (Figure 1D), which has two Egr-1 promoter-proximal SREs upstream of the minimal promoter. Figure 1E shows that transcription of the chromatin-integrated Egr-1.SRE.luc reporter gene is significantly induced in HIRcB cells stimulated with insulin. Therefore, the SRE can be considered to function as an insulin-responsive element.

Figure 1. Increased Egr-1 promoter activity and Egr-1 expression in insulin-stimulated HIRcB cells. (Thiel G, et al., 2021)