BACKGROUND AND PURPOSETransient receptor potential-3 (TRPM3) channels function as Ca 2+ permeable cation channels. While the natural ligands for these channels are still unknown, several compounds have been described that either activate or inhibit TRPM3 channel activity. EXPERIMENTAL APPROACHWe assessed TRPM3-mediated gene transcription, which relies on the induction of intracellular signalling to the nucleus following activation of TRPM3 channels. Activator protein-1 (AP-1) and Egr-1-responsive reporter genes were integrated into the chromatin of the cells. This strategy enabled us to analyse gene transcription of the AP-1 and Egr-1-responsive reporter genes that were packed into an ordered chromatin structure. KEY RESULTSThe neurosteroid pregnenolone sulfate strikingly up-regulated AP-1 and Egr-1 transcriptional activity, while nifedipine and D-erythro-sphingosine, also putative activators of TRPM3 channels, exhibited either no or TRPM3-independent effects on gene transcription. In addition, pregnenolone sulfate robustly enhanced the transcriptional activation potential of the ternary complex factor Elk-1. Pregnenolone sulfate-induced activation of gene transcription was blocked by treatment with mefenamic acid and, to a lesser extent, by the polyphenol naringenin. In contrast, progesterone, pregnenolone and rosiglitazone reduced AP-1 activity in the cells, but had no inhibitory effect on Egr-1 activity in pregnenolone sulfate-stimulated cells. CONCLUSION AND IMPLICATIONSPregnenolone sulfate is a powerful activator of TRPM3-mediated gene transcription, while transcription is completely inhibited by mefenamic acid in cells expressing activated TRPM3 channels. Both compounds are valuable tools for further investigating the biological functions of TRPM3 channels. LINKED ARTICLESThis article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx

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LDL-cholesterol (LDL-C) is a causal pathogenic factor in atherosclerosis. Monoclonal anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralizing antibodies are novel potent LDL-lowering drugs which reduce cardiovascular events. To characterize their effect on atherogenesis, APOE*3Leiden.CETP mice were fed a high cholesterol/high fat diet (WTD) or normal chow (NC) for 18 weeks. Mice on WTD were injected with the human anti-PCSK9 antibody mAb1 (PL-45134, 10 mg*kg −1 s.c.) or 0.9% saline every 10 days. PCSK9 inhibition decreased total cholesterol in serum of APOE*3Leiden.CETP mice and prevented the development of atherosclerosis. The plaque area in the aortic root was reduced by half and macrophage infiltration determined by Ly6c and Mac-3 staining was ameliorated. PCSK9 inhibition decreased markers of inflammation in mononuclear cells ( Il-6, Tnfa mRNA), and in serum (CXCL-1,-10,-13; complement factor C5a) compared to control WTD fed animals. The number of circulating Sca-1/VEGF-R2 positive endothelial progenitor cells of the peripheral blood and spleen-derived diLDL/lectin double positive circulating angiogenic cells was increased. To conclude, the PCSK9-mediated anti-atherosclerotic effect involves the upregulation of pro-regeneratory endothelial progenitor cells, a reduction of inflammation and change of plaque composition.

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BACKGROUND AND PURPOSE The rise in intracellular Ca 2+ stimulates the expression of the transcription factor c-Fos. Depending on the mode of entry of Ca 2+ into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca 2+ influx via activation of transient receptor potential melastatin-3 (TRPM3) channels with enhanced c-Fos expression. EXPERIMENTAL APPROACH Transcription of c-Fos promoter/reporter genes that were integrated into the chromatin via lentiviral gene transfer was analysed in HEK293 cells overexpressing TRPM3. The transcriptional activation potential of c-Fos was measured using a GAL4-c-Fos fusion protein. KEY RESULTS The signalling pathway connecting TRPM3 stimulation with enhanced c-Fos expression requires the activation of MAP kinases. On the transcriptional level, three Ca 2+-responsive elements, the cAMP-response element and the binding sites for the serum response factor (SRF) and AP-1, are essential for the TRPM3-mediated stimulation of the c-Fos promoter. Ternary complex factors are additionally involved in connecting TRPM3 stimulation with the up-regulation of c-Fos expression. Stimulation of TRPM3 channels also increases the transcriptional activation potential of c-Fos. CONCLUSIONS AND IMPLICATIONS Signalling molecules involved in connecting TRPM3 with the c-Fos gene are MAP kinases and the transcription factors CREB, SRF, AP-1 and ternary complex factors. As c-Fos constitutes, together with other basic region leucine zipper transcription factors, the AP-1 transcription factor complex, the results of this study explain TRPM3-induced activation of AP-1 and connects TRPM3 with the biological functions regulated by AP-1. Abbreviations AP-1, activator protein-1; bZIP, basic region leucine zipper; CREB, cAMP-response element-binding protein; SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor; TPA, phorbol 12-myristate 13-acetate; TRP, transient receptor potential BJP British Journal of Pharmacology