br Materials and methods br Results

Materials and methods
Results
Discussion Recently, we showed that stimulation of TRPM3 and TRPC6 channels triggers a signaling cascade leading to the activation of AP-1 [11], [12], [13], [15]. The objective of this study was to investigate whether stimulation of TRPV1 channels leads to an activation of AP-1 as well. AP-1 is a dimeric transcription factor composed of bZIP proteins of the Fos, Jun, and ATF (activating transcription factor) family of transcription factors. Originally, AP-1 was described as a heterodimer of c-Jun and c-Fos [40]. AP-1 is a convergence point for many intracellular signaling cascades, regulating many biological functions, including the regulation of proliferation, transformation, differentiation, and apoptosis [44]. To measure AP-1 activity in the cells, an AP-1-responsive reporter gene was used as suggested by Karin [16] which was integrated into the chromatin to ensure that it was packed into an ordered nucleosomal structure. The results of this study show that stimulation with the TRPV1 ligands capsaicin or RTX activated the AP-1 transcription factor in a TRPV1-dependent manner. NADA exhibited a TRPV1-dependent and a TRPV1-independent activation of AP-1. Endovanilloids such as NADA are not TRPV1-specific ligands. Rather, these compounds are promiscuous, as shown by the observation that NADA activates CB1 cannabinoid receptors and also shows TRPV1 and CB1 receptor-independent activities [45], [46]. The nature of the NADA receptor in HEK293 Valifenalate is not yet known. Experiments involving overexpression of regulator of G protein signaling-2 (RGS2) were negative (T.M. Backes, G.Thiel, unpublished observations), indicating that receptors coupled to the Gαq protein are not involved in NADA signaling. The activation of AP-1 in TRPV1 expressing cells was not the result of ligand toxicity as observed for cortical neurons [36], [37]. Thus, neurons are much more sensitive in terms of capsaicin-induced toxicity in comparison to HEK293-derived H2C1 cells. TRPV1 functions as a Ca2+ permeable cation channel [3]. Accordingly, the influx of Ca2+ into the cells and the subsequent rise in the intracellular Ca2+ concentration has been frequently used as an indicator of activated TRPV1 channels [3], [5]. Ca2+ imaging experiments revealed that stimulation of H2C1 cells expressing TRPV1 channels with either capsaicin, RTX, or NADA increased the intracellular Ca2+ concentration. While stimulation of HEK293 cells with either capsaicin or RTX induced no or only marginal changes in the intracellular Ca2+ concentration, stimulation with NADA led to a rise in intracellular [Ca2+]i. This observation explains the TRPV1-independent activation of AP-1 in NADA-stimulated HEK293 cells, which is most likely based on a TRPV1-independent influx of Ca2+ following stimulation of HEK293 cells with NADA. Naturally, higher intracellular Ca2+ concentrations were measured in H2C1 cells, based on TRPV1-dependent and TRPV1-independent influx of Ca2+. A rise in cytoplasmic Ca2+ levels, induced by stimulating either Gαq-coupled receptors, voltage or ligand-activated Ca2+ channels, activates the protein kinase ERK1/2 [13], [35], [47], [48]. The results of this study reveal that stimulation of TRPV1 channels with capsaicin also leads to a rapid phosphorylation and activation of ERK1/2. Pharmacological experiments, using the compound PD98059 as a tool for a selective inhibition of the ERK1/2 signaling pathway, clearly showed that the activation of ERK is essential to connect TRPV1 stimulation with AP-1 activation. Moreover, expression experiments involving the nuclear phosphatase MKP-1 revealed that an activated nuclear ERK is needed for stimulation of AP-1 in H2C1 cells. MKP-1 may function as a nuclear shut-off device that interrupts the signaling cascades initiated by capsaicin stimulation of TRPV1 channels. Together, these data identified ERK1/2 as the signal transducer that connects activated TRPV1 channels with AP-1-regulated gene transcription.