Bitter taste signaling mediated by Tas2r144 is down-regulated by 17β-estradiol and progesterone in the rat choroid plexus
Introduction
The blood-cerebrospinal fluid barrier (BCSFB) is composed by the choroid plexuses (CPs), formed by a monolayer of epithelial cells that lie over a highly vascularized stroma, floating within the brain ventricles. The CPs epithelial cells (CPEC) secrete most of the cerebrospinal fluid (CSF) (Damkier et al., 2013) and help to maintain a stable extracellular environment in the brain. Besides the barrier function itself, the CPs supply nutrients and hormones to the CSF and brain, ensure immune surveillance (Schwartz and Baruch, 2014), contribute with growth factors to the CSF that are essential for neurogenesis (Falcao et al., 2012) and more recent evidences show that the CPs also work as a peripheral clock and are a source of melatonin comparable to the pineal gland (Myung et al., 2018; Quintela et al., 2018, 2015). Chemical surveillance is another essential function of the CPs that includes the clearance and detoxification of toxic compounds (Ghersi-Egea et al., 2018b, 2018a; Johanson et al., 2011; Strazielle and Ghersi-Egea, 2016), as amyloid clearance (Pahnke et al., 2014). However, upstream regulators of this chemical surveillance system are still largely unknown. We found a large number of transcripts encoding chemosensory receptors such as olfactory and taste receptors, and demonstrated that the essential components of olfactory and taste signaling pathways are expressed and operational in the CPs (Gonçalves et al., 2016; Tomás et al., 2016). The discovery of functional taste receptors and their downstream effectors in the CP, suggests that the taste signaling pathway in the CP may be crucial for its function in the assessment of the CSF and/or blood composition, to deploy detoxification pathways and molecular trafficking within CP cells.
Taste receptors that bind sweet (Tas1R2/Tas1R3) umami (Tas1R1/Tas1R3) and bitter (Tas2rs) compounds belong to the G Protein-Coupled Receptor (GPCR) family. Upon ligand-binding they undergo a conformational change and activate a taste-specific heterotrimeric G-protein gustducin which in turn, activates phospholipase C-beta 2 (Plcb2) to produce inositol 1,4,5-trisphosphate (IP3). The IP3 opens receptor type 3 ion channels (IP3R3), provoking an increase in intracellular Ca2+ levels that will activate the transient receptor potential cation channel, subfamily M, member 5 (Trpm5), that eventually depolarizes the cell (Chandrashekar et al., 2006). The CPs express sweet and umami receptors, and 32 of the 36 bitter receptors (Tas2rs) expressed in rat, as well as the downstream effector proteins: α-Gust, Plcb2, IP3R3 and Trpm5 (Quintela et al., 2013; Tomás et al., 2016).
The CPs are multifunctional sex hormone targets (Quintela et al., 2013) containing sex hormones receptors such as progesterone receptor (PR) (Quadros et al., 2007), alpha and beta estrogen receptor (ER) (Hong-Goka and Chang, 2004) and androgen receptor (Alves et al., 2009).
Interestingly, the analysis of the effects of the sex hormone background in the rat CPs transcriptome has shown that the taste signaling pathway is one of the top five pathways regulated by female sex hormones (Quintela et al., 2013) with Tas2rs genes Tas2r109 (taste receptor, type 2, member 109), Tas2r124 (taste receptor, type 2, member 124), Tas2r134 (taste receptor, type 2, member 134), Tas2r137 (taste receptor, type 2, member 137) and Tas2r144 (taste receptor, type 2, member 144) expressed at higher levels (at least two-fold) in the CPs of ovariectomized (OVX) female Wistar rats when compared to sham females (Quintela et al., 2013; Santos et al., 2017).
The taste signaling pathway was once thought to be exclusive of the oral cavity, but over the past decade, several studies clearly demonstrated different roles of this pathway. Notably, Tas2rs are expressed in several extra-oral organs, including the respiratory, urinary, digestive and immune systems, as well as in different regions of the brain, with different functional implications according to their localization (Foster et al., 2014; Lu et al., 2017; Shaik et al., 2016; Tomás et al., 2016). In barrier organs such as the airways and the gastrointestinal tract, the activation of Tas2rs triggers different types of protective responses in the organism. For instance, Tas2rs activation in the airways, evoke changes in the respiratory rate (Finger et al., 2003; Krasteva et al., 2011; Tizzano et al., 2010), regulates innate immunity (Hariri et al., 2017; Lee and Cohen, 2015a, 2015b; Lee et al., 2014; Tizzano et al., 2010), increase ciliary motility (Shah et al., 2009) and promote bronchodilation (Camoretti-Mercado et al., 2015; Deshpande et al., 2010; Lifshitz et al., 2013; Robinett et al., 2014; Tan and Sanderson, 2014). In the gastrointestinal tract, Tas2rs regulate nutrient transporter expression and nutrient uptake, the release of gut hormones and neurotransmitters involved in the regulation of energy and glucose homeostasis controlling food intake, gastric motility and ion secretion, promoting fluid secretion by the epithelium to flush the noxious irritant compounds out of the body (Avau and Depoortere, 2015; Chen et al., 2006; Jeon et al., 2008, 2011; Kaji et al., 2009; Rozengurt, 2006; Rozengurt et al., 2006).
Although Tas2rs functions have been overlooked in the brain, altered levels of Tas2rs have been found in the frontal cortex in Parkinson's disease (Garcia-Esparcia et al., 2013), in the entorhinal and frontal cortex in Alzheimer's disease, in the frontal cortex in terminal stages of Progressive Supranuclear Palsy, and in the frontal cortex and cerebellum in Creutzfeldt-Jakob disease subtypes (Ansoleaga et al., 2013). However, there are no studies defining a role for Tas2rs on the onset of central nervous system (CNS) pathologies. A number of these CNS pathologies such as Parkinson's disease (Jurado-Coronel et al., 2017), Alzheimer's disease (Li and Singh, 2014), or multiple sclerosis (Hanamsagar and Bilbo, 2016) differ between sexes in their clinical presentation, prevalence, symptoms and prognosis. Most of these pathologies present compromised CSF clearance mechanisms, which may be related to altered chemical surveillance of the CSF and/or blood (Marques et al., 2016). Understanding the regulation of taste transduction by sex hormones in the CPs may thus elucidate the mechanisms behind the chemical surveillance capacity of this organ, contributing to clarify how sex-differences may relate to CNS disease susceptibility and drug resistance.
This study focused on the regulation of key components and functioning of the bitter taste signaling pathway by female sex hormones, 17β-estradiol (E2) and progesterone (P4). We selected two taste receptors upregulated by ovariectomy, as determined by cDNA microarrays, Tas2r109 and Tas2r144, and the effector proteins Plcb2 and Trpm5 (Quintela et al., 2013). The effects of E2 and P4 on the regulation of these genes in rat CPs, and the molecular mechanisms behind their regulation were analyzed in terms of gene expression (RT-qPCR) and functionality (calcium imaging) after bitter taste signaling activation.
Section snippets
Animals
All the experiments on rats were handled in compliance with the NIH guidelines, and the European Union rules for the care and handling of laboratory animals (Directive, 2010/63/EU). Adult female Wistar rats (8–10 week old) were used to monitor the effect of female sex hormones on the expression of Tas2r109, Tas2r144, Plcb2 and Trpm5 genes. All the animals were housed in appropriate cages at constant room temperature (Sealsafe Individual Ventilated Cages Blue Line – 1291H, coupled to an Air
Ovariectomy up-regulates the expression of taste-related genes
The effects of female sex hormones on the expression of the taste-related genes were analyzed by real time RT-qPCR in the CPs of OVX and sham female rats. There was a clear increase of the expression of Tas2r109 (2.5 fold), Tas2r144 (2.2 fold), Plcb2 (2.2 fold) and Trpm5 (1.3 fold) genes (Fig. 1) in the CPs of OVX rats two weeks after ovariectomy, compared to sham-operated animals.
These expression differences have a similar magnitude to those obtained by analysis of our cDNA microarrays data (
Discussion
In a recent study, we reported the expression of genes of the canonical taste signaling pathway in CPs, and demonstrated its functionality by observing responses in primary cultures of CPEC to the bitter compound D-Salicin (Tomás et al., 2016). In addition, data from a CPs microarray study showed that the decline of hormone levels in female rats upon OVX clearly induced up-regulation of genes of this pathway (Quintela et al., 2013), including the bitter taste receptors Tas2r109, Tas2r124,
Conclusion
In conclusion, our findings indicate a functional regulation of the bitter taste signaling in CPs by female sex hormones. In particular, we show that E2 and P4 down-regulate the expression of Tas2r109, Tas2r144, Plcb2 and Trpm5 genes. These E2 and P4 effects seem to be mediated by ERs (and/or mER) and by the mPR respectively. We also report that the functional bitter taste receptor, Tas2r144, that responds to DB stimuli in CP cells, is inhibited by female sex hormones. The taste chemosensory
Conflicts of interest
The authors declare no competing interest
Acknowledgements
This work was supported by the Portuguese Society for Endocrinology, Diabetes & Metabolism (Bolsa para projeto de investigação científica em endocrinologia 2018), by the Portuguese Foundation for Science and Technology (FCT, Portugal – http://www.fct.pt) project grants (PTDC/SAU-NEU/114800/2009, project UID/Multi/04326/2013, project UID/Multi/00709/2013 and UID/Multi/00709/2019), and FEDER funds through the POCI –COMPETE 2020 – Operational Program Competitiveness and Internationalization in
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