Trace amine-associated receptors (TAARs) belong to the class A G-protein-coupled receptors (GPCR) and are evolutionary related to aminergic receptors. TAARs have been identified to mediate effects of trace amines. TAAR1 signaling is mainly mediated via activation of the Gs/adenylyl cyclase pathway. In addition to classical trace amines, TAAR1 can also be activated by the thyroid hormone derivative 3-iodothyronamine (3-T1AM). Pharmacological doses of 3-T1AM induced metabolic and anapyrexic effects, which might be centrally mediated in the hypothalamus in rodents. However, the observed anapyrexic effect of 3-T1AM persists in Taar1 knock-out mice which raises the question whether further GPCRs are potential targets for 3-T1AM and mediate the observed physiological effect. Anapyrexia has been observed to be related to action on aminergic receptors such as the serotonin receptor 1b (5-HT1b). This receptor primarily activates the Gi/o mediated pathway and PLC signaling through the Gβγ of Gi/o. Since the expression profiles of TAAR1 and 5-HT1b overlap, we questioned whether 3-T1AM may activate 5-HT1b. Finally, we also evaluated heteromerization between these two GPCRs and tested signaling under co-expressed conditions. In this study, we showed, that 3-T1AM can induce Gi/o signaling through 5-HT1b in a concentration of 10 μM. Strikingly, at 5-HT1b the ligand 3-T1AM only activates the Gi/o mediated reduction of cAMP accumulation, but not PLC activation. Co-stimulation of 5-HT1b by both ligands did not lead to additive or synergistic signaling effects. In addition, we confirmed the capacity for heteromerization between TAAR1 and 5-HT1b. Under co-expression of TAAR1 and HTR1b, 3-T1AM action is only mediated via TAAR1 and activation of 5-HT1b is abrogated. In conclusion, we found evidence for 5-HT1b as a new receptor target for 3-T1AM, albeit with a different signaling effect than the endogenous ligand. Altogether, this indicates a complex interrelation of signaling effects between the investigated GPCRs and respective ligands.
Functional in vitro models emulating the physiological processes of human organ formation are invaluable for future research and the development of regenerative therapies. Here, a developmentally inspired approach is pursued to reproduce fundamental steps of human tooth organogenesis in vitro using human dental pulp cells. Similar to the in vivo situation of tooth initiating mesenchymal condensation, a 3D self-organizing culture was pursued resulting in an organoid of the size of a human tooth germ with odontogenic marker expression. Furthermore, the model is capable of epithelial invagination into the condensed mesenchyme, mimicking the reciprocal tissue interactions of human tooth development. Comprehensive transcriptome analysis revealed activation of well-studied as well as rather less investigated signaling pathways implicated in human tooth organogenesis, such as the Notch signaling. Early condensation in vitro revealed a shift to the TGFß signal transduction pathway and a decreased RhoA small GTPase activity, connected to the remodeling of the cytoskeleton and actin-mediated mechanotransduction. Therefore, this in vitro model of tooth development provides a valuable model to study basic human developmental mechanisms.
3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells.
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