ACKR3 in olfactory glia cells shapes the immune defense of the olfactory mucosa.

Barrier-forming olfactory glia cells, termed sustentacular cells, play important roles for immune defense of the olfactory mucosa, for example as entry sites for SARS-CoV-2 and subsequent development of inflammation-induced smell loss. Here we demonstrate that sustentacular cells express ACKR3, a chemokine receptor that functions both as a scavenger of the chemokine CXCL12 and as an activator of alternative signaling pathways. Differential gene expression analysis of bulk RNA sequencing data obtained from WT and ACKR3 conditional knockout mice revealed upregulation of genes involved in immune defense. To map the regulated genes to the different cell types of the olfactory mucosa, we employed biocomputational methods utilizing a single-cell reference atlas. Transcriptome analysis, PCR and immunofluorescence identified up-regulation of NF-κB-related genes, known to amplify inflammatory signaling and to facilitate leukocyte transmigration, in the gliogenic lineage. Accordingly, we found a marked increase in leukocyte-expressed genes and confirmed leukocyte infiltration into the olfactory mucosa. In addition, lack of ACKR3 led to enhanced expression and secretion of early mediators of immune defense by Bowman's glands. As a result, the number of apoptotic cells in the epithelium was decreased. In conclusion, our research underlines the importance of sustentacular cells in immune defense of the olfactory mucosa. Moreover, it identifies ACKR3, a druggable G protein-coupled receptor, as a promising target for modulation of inflammation-associated anosmia.

Chemokine signaling is required for homeostatic and injury-induced neurogenesis in the olfactory epithelium.

The olfactory epithelium (OE) possesses unique lifelong neuroregenerative capacities and undergoes constitutive neurogenesis throughout mammalian lifespan. Two populations of stem cells, frequently dividing globose basal cells (GBCs) and quiescent horizontal basal cells (HBCs), readily replace olfactory neurons throughout lifetime. Although lineage commitment and neuronal differentiation of stem cells has already been described in terms of transcription factor expression, little is known about external factors balancing between differentiation and self-renewal. We show here that expression of the CXC-motif chemokine receptor 4 (CXCR4) distinguishes both types of stem cells. Extensive colocalization analysis revealed exclusive expression of CXCR4 in proliferating GBCs and their neuronal progenies. Moreover, only neuronal lineage cells were derived from CXCR4-CreER-tdTomato reporter mice in the OE. Furthermore, Cre-tdTomato mice specific for HBCs (Nestin+ and Cytokeratin14+) did not reduce CXCR4 expression when bred to mice bearing floxed CXCR4 alleles, and did not show labeling of the neuronal cells. CXCR4 and its ligand CXCL12 were markedly upregulated upon induction of GBC proliferation during injury-induced regeneration. in vivo overexpression of CXCL12 did downregulate CXCR4 levels, which results in reduced GBC maintenance and neuronal differentiation. We proved that these effects were caused by CXCR4 downregulation rather than over-activation by showing that the phenotypes of CXCL12-overexpressing mice were highly similar to the phenotypes of CXCR4 knockout mice. Our results demonstrate functional CXCR4 signaling in GBCs regulates cell cycle exit and neural differentiation. We propose that CXCR4/CXCL12 signaling is an essential regulator of olfactory neurogenesis and provide new insights into the dynamics of neurogenesis in the OE.

Functional Characterization of the Odorant Receptor 51E2 in Human Melanocytes.

Olfactory receptors, which belong to the family of G-protein-coupled receptors, are found to be ectopically expressed in non-sensory tissues mediating a variety of cellular functions. In this study we detected the olfactory receptor OR51E2 at the transcript and the protein level in human epidermal melanocytes. Stimulation of primary melanocytes with the OR51E2 ligand ß-ionone significantly inhibited melanocyte proliferation. Our results further showed that ß-ionone stimulates melanogenesis and dendritogenesis. Using RNA silencing and receptor antagonists, we demonstrated that OR51E2 activation elevated cytosolic Ca(2+) and cAMP, which could mediate the observed increase in melanin synthesis. Co-immunocytochemical stainings using a specific OR51E2 antibody revealed subcellular localization of the receptor in early endosomes associated with EEA-1 (early endosome antigen 1). Plasma membrane preparations showed that OR51E2 protein is present at the melanocyte cell surface. Our findings thus suggest that activation of olfactory receptor signaling by external compounds can influence melanocyte homeostasis.

Functional expression of olfactory receptors in human primary melanoma and melanoma metastasis.

We identified the olfactory receptor 51E2 in human melanoma and have measured both OR51E2 mRNA and protein expression in melanoma tissue sections. qPCR analysis revealed that the receptor is upregulated in melanoma cells compared to normal melanocytes, indicating that OR51E2 may play a role in early melanoma development and progression. Activation of endogenous OR51E2 in cultured cells derived from metastatic and vertical-growth phase (VGP) by its ligand ß-ionone results in an increase in the intracellular Ca2+ concentration. RNAi experiments showed that the ß-ionone-induced Ca2+ signal depends on the activation of OR51E2. Furthermore, OR51E2 activation inhibits the growth of VGP melanoma cells via apoptotic processes. Cell motility assays revealed that treatment with ß-ionone decreases the migration of VGP melanoma cells. Overall, our data demonstrates that OR51E2 is involved in the regulation of cell proliferation and migration, suggesting that it may serve as a novel target for melanoma therapy.

NHERF1 in Microvilli of Vomeronasal Sensory Neurons.

In most mammals, the vomeronasal system detects a variety of (semio)chemicals that mediate olfactory-driven social and sexual behaviors. Vomeronasal chemosensation depends on G protein-coupled receptors (V1R, V2R, and FPR-rs) that operate at remarkably low stimulus concentrations, thus, indicating a highly sensitive and efficient signaling pathway. We identified the PDZ domain-containing protein, Na+/H+ exchanger regulatory factor-1 (NHERF1), as putative molecular organizer of signal transduction in vomeronasal neurons. NHERF1 is a protein that contains 2 PDZ domains and a carboxy-terminal ezrin-binding domain. It localizes to microvilli of vomeronasal sensory neurons and interacts with V1Rs. Furthermore, NHERF1 and Gαi2 are closely colocalized. These findings open up new aspects of the functional organization and regulation of vomeronasal signal transduction by PDZ scaffolding proteins.

Quantitative phosphoproteomics reveals the protein tyrosine kinase Pyk2 as a central effector of olfactory receptor signaling in prostate cancer cells.

The prostate-specific G-protein-coupled receptor 1 (PSGR1) is an olfactory receptor specifically expressed in the prostate gland. PSGR1 expression is elevated both in benign prostatic hyperplasia tissue and in prostate cancer. Stimulation of PSGR1 by the odorant ß-ionone leads to an increase in the intracellular Ca(2+) concentration, activation of mitogen-activated protein (MAP) kinases and a decrease in prostate cancer cell proliferation. To further extend our knowledge about PSGR1 signaling in prostate cancer cells, we performed a quantitative phosphoproteomics study using stable isotope labeling by amino acids in cell culture and mass spectrometry. We report 51 differentially regulated phosphorylation sites in 24 proteins with functions in cytoskeletal remodeling, signaling and ion transport. Activation of PSGR1 evoked an increase in intracellular pH mediated by the sodium/hydrogen exchanger NHE1. Furthermore, we report the protein tyrosine kinase Pyk2 as a central effector of PSGR1 signaling cascades in LNCaP cells. Our data show that phosphorylation of p38 MAP kinase is triggered by Pyk2. In addition, we confirmed dephosphorylation of the tumor suppressor protein N-myc downstream regulated gene 1 (NDRG1) at Ser330 downstream of Pyk2. Since NDRG1 impacts oncogenic signaling pathways interfering with tumor progression, we suggest that the Pyk2-NDRG1 axis is possibly involved in conveying the anti-proliferative effect of ß-ionone in prostate cancer cells. This article is part of a Special Issue entitled: Medical Proteomics.

The scaffold protein MUPP1 regulates odorant-mediated signaling in olfactory sensory neurons.

The olfactory signal transduction cascade transforms odor information into electrical signals by a cAMP-based amplification mechanism. The mechanisms underlying the very precise temporal and spatial organization of the relevant signaling components remains poorly understood. Here, we identify, using co-immunoprecipitation experiments, a macromolecular assembly of signal transduction components in mouse olfactory neurons, organized through MUPP1. Disruption of the PDZ signaling complex, through use of an inhibitory peptide, strongly impaired odor responses and changed the activation kinetics of olfactory sensory neurons. In addition, our experiments demonstrate that termination of the response is dependent on PDZ-based scaffolding. These findings provide new insights into the functional organization, and regulation, of olfactory signal transduction.

Elevated Cytosolic Cl- Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons.

In rodents, the vomeronasal system controls social and sexual behavior. However, several mechanistic aspects of sensory signaling in the vomeronasal organ remain unclear. Here, we investigate the biophysical basis of a recently proposed vomeronasal signal transduction component-a Ca(2+)-activated Cl(-) current. As the physiological role of such a current is a direct function of the Cl(-) equilibrium potential, we determined the intracellular Cl(-) concentration in dendritic knobs of vomeronasal neurons. Quantitative fluorescence lifetime imaging of a Cl(-)-sensitive dye at the apical surface of the intact vomeronasal neuroepithelium revealed increased cytosolic Cl(-) levels in dendritic knobs, a substantially lower Cl(-) concentration in vomeronasal sustentacular cells, and an apparent Cl(-) gradient in vomeronasal neurons along their dendritic apicobasal axis. Together, our data provide a biophysical basis for sensory signal amplification in vomeronasal neuron microvilli by opening Ca(2+)-activated Cl(-) channels.

Co-expression of anoctamins in cilia of olfactory sensory neurons.

Vertebrates can sense and identify a vast array of chemical cues. The molecular machinery involved in chemodetection and transduction is expressed within the cilia of olfactory sensory neurons. Currently, there is only limited information available on the distribution and density of individual signaling components within the ciliary compartment. Using super-resolution microscopy, we show here that cyclic-nucleotide-gated channels and calcium-activated chloride channels of the anoctamin family are localized to discrete microdomains in the ciliary membrane. In addition to ANO2, a second anoctamin, ANO6, also localizes to ciliary microdomains. This observation, together with the fact that ANO6 and ANO2 co-localize, indicates a role for ANO6 in olfactory signaling. We show that both ANO2 and ANO6 can form heteromultimers and that this heteromerization alters the recombinant channels' physiological properties. Thus, we provide evidence for interaction of ANO2 and ANO6 in olfactory cilia, with possible physiological relevance for olfactory signaling.

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding.

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.

Amiloride derivatives are effective blockers of insect odorant receptors.

Heteromeric insect odorant receptors (ORs) form ligand-activated nonselective cation channels in recombinant expression systems. We performed a pharmacological characterization of Drosophila melanogaster and Bombyx mori ORs expressed in the Xenopus laevis oocyte expression system and characterized them using the 2-electrode voltage clamp. We identified amiloride derivatives as high-affinity blockers, which inhibit the ion current through the channel in a low micromolar range. For the heteromeric Drosophila Or47a + DmelOrco receptor, the potency sequence (IC(50)) is HMA [5-(N,N-hexamethylene)amiloride] (3.9 µM), MIA [5-(N-methyl-N-isobutyl)amiloride] (11.0 µM), and DMA [5-(N,N-dimethyl)amiloride] (113.3 µM). Amiloride itself is nearly ineffective. Other tested insect ORs (Drosophila Or49b + DmelOrco, B. mori BmorOr1 + BmorOrco) were blocked in a similar fashion suggesting that the amiloride derivatives were potential general blockers of all receptor combinations. Our results suggest that pyrazine derivatives of amiloride are useful probes to study the mechanism of chemosensory transduction in insects in more detail.

G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation.

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.

Chemosensory Ca2+ dynamics correlate with diverse behavioral phenotypes in human sperm.

In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca(2+) signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca(2+) dynamics, and the relation between a distinct Ca(2+) signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca(2+) responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca(2+) signaling patterns with unique spatiotemporal dynamics. These distinct Ca(2+) dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.

Purinergic signalling mobilizes mitochondrial Ca²âº in mouse Sertoli cells.

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Molecular evolution of a chordate specific family of G protein-coupled receptors.

BACKGROUND: Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. RESULTS: We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. CONCLUSIONS: GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become chordates.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

Go α is involved in sugar perception in Drosophila.

Detection of chemical compounds in food sources is based on the activation of 7 transmembrane gustatory receptors (GRs) in mammals and in insects such as Drosophila, although the receptors are not conserved between the classes. Different combinations of Drosophila GRs are involved in the detection of sugars, but the activated signaling cascades are largely unknown. Because 7 transmembrane receptors usually couple to G-proteins, we tried to unravel the intracellular signaling cascade in taste neurons by screening heterotrimeric G-protein mutant flies for gustatory deficits. We found the subunit Goα to be involved in feeding behavior and cell excitability by different transgenic and pharmacological approaches. Goα is involved in the detection of sucrose, glucose, and fructose, but not with trehalose and maltose. Our studies reveal that Goα plays an important role in the perception of some sweet tastants. Because the perception of other sweet stimuli was not affected by mutations in Goα, we also found strong indication for the existence of multiple signaling pathways in the insect gustatory system.

Tmem16b is specifically expressed in the cilia of olfactory sensory neurons.

Calcium-activated chloride channels (CaCCs) are involved in many physiological processes, including sensory signal transduction, but only little is known to date about their structure and function. We performed a proteome analysis of the olfactory epithelium (OE) membrane proteome and identified so far uncharacterized membrane proteins as candidate channels. One of the most abundant membrane proteins in olfactory sensory neurons (OSNs) was Tmem16b, a member of a recently identified family of CaCCs. In addition to former studies performed on Tmem16b, we show here that Tmem16b expression is highly specific for the OE, in contrast to the closely related Tmem16a, which shows a broad expression pattern in secretory epithelial cells. Native Tmem16b is localized in the cilia of the OSNs, which is in agreement with previous electrophysiological recordings.

Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster.

Despite increasing knowledge about dimerization of G-protein-coupled receptors, nothing is known about dimerization in the largest subfamily, odorant receptors. Using a combination of biochemical and electrophysiological approaches, we demonstrate here that odorant receptors can dimerize. DOR83b, an odorant receptor that is ubiquitously expressed in olfactory neurons from Drosophila melanogaster and highly conserved among insect species, forms heterodimeric complexes with other odorant-receptor proteins, which strongly increases their functionality.

Mimicking the olfactory system for the classification of chemical data.

Various computational techniques have been used in pharmacological research to classify chemical compounds based on their physicochemical properties and putative biological activity. The recent publication by Schmuker and Schneider describes a new approach for the processing and classification of chemical data. Their study was motivated by nature's solution for detection and discrimination of chemical data, which is manifested in the olfactory systems of vertebrates and invertebrates.