AP1 Luciferase Reporter Lentivirus

Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca2+ ions into cells and results in an increase in intracellular Ca2+ concentration, which activates the activator protein 1 (AP-1) transcription factor. Here, the researchers show that expression of a constitutively active mutant of the Ca2+/calmodulin-dependent protein phosphatase calcineurin attenuates pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Similarly, expression of the regulatory B subunit of calcineurin reduces AP-1 activity in cells following stimulation of TRPM3 channels. MAP kinase phosphatase 1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, the researchers show that pregnenolone sulfate-induced TRPM3 stimulation triggers phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Stimulation of ERK1/2 is essential for AP-1 activation in cells expressing stimulated TRPM3 channels. ERK1/2 is required for activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor and regulates c-Fos promoter activity. Altogether, the data suggest that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators in the signaling cascade linking TRPM3 channel stimulation to increased AP-1-regulated transcription.

Calcineurin consists of two polypeptides, the catalytic calcineurin A subunit and the regulatory B subunit. Lentiviral gene transfer was used to express a constitutively active calcineurin A mutant, △CnA, which lacks the calmodulin binding site and the C-terminal autoinhibitory domain and does not require Ca2+ ions for activation. The domain structure of wild type and mutant calcineurin A is depicted in Fig. 1A. As a sensor to measure AP-1 activity, researchers used lentivirus containing the AP-1-responsive collagenase promoter/luciferase reporter gene. HEK293 cells containing a tetracyclineresponsive TRPM3 expression cassette were infected with a lentivirus containing the luciferase gene under the control of the collagenase promoter (Coll.luc). In addition, cells were either mock infected or infected with a lentivirus encoding the truncated calcineurin A mutant △CnA. Cells were treated with tetracycline to induce TRPM3 expression. Fig. 1B shows that expression of △CnA significantly reduced the activation of AP-1 in HEK293 cells expressing stimulated TRPM3 channels. Similar results were obtained after expressing △CnA-EGFP fusion protein in cells (Figure 1C, 1D). Visualization of EGFP expression showed that the fusion protein was mainly expressed in the cytosol (Figure 1E).

Figure 1. Expression of a constitutively active mutant of calcineurin A interrupts the signaling cascade connecting TRPM3 channels with the AP-1 transcription factor. (Lesch A, Rössler O G, Thiel G., 2017)