TPC Functions in the Immune System.

Two-pore channels (TPCs) are novel intracellular cation channels, which play a key role in numerous (patho-)physiological and immunological processes. In this chapter, we focus on their function in immune cells and immune reactions. Therefore, we first give an overview of the cellular immune response and the partaking immune cells. Second, we concentrate on ion channels which in the past have been shown to play an important role in the regulation of immune cells. The main focus is then directed to TPCs, which are primarily located in the membranes of acidic organelles, such as lysosomes or endolysosomes but also certain other vesicles. They regulate Ca2+ homeostasis and thus Ca2+ signaling in immune cells. Due to this important functional role, TPCs are enjoying increasing attention within the field of immunology in the last few decades but are also becoming more pertinent as pharmacological targets for the treatment of pro-inflammatory diseases such as allergic hypersensitivity. However, to uncover the precise molecular mechanism of TPCs in immune cell responses, further molecular, genetic, and ultrastructural investigations on TPCs are necessary, which then may pave the way to develop novel therapeutic strategies to treat diseases such as anaphylaxis more specifically.

TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity.

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.

Physiological Temperature Changes Fine-Tune ß 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro.

The chemical warfare agent sulfur mustard (SM) alkylates a multitude of biomacromolecules including DNA and proteins. Cysteine residues and nucleophilic nitrogen atoms in purine DNA bases are typical targets of SM but potentially every nucleophilic structure may be alkylated by SM. In the present study, we analyzed potential SM-induced alkylation of glucocorticoid (GC) hormones and functional consequences thereof. Hydrocortisone (HC), the synthetic betamethasone (BM) and dexamethasone (DEX) were chosen as representative GCs. Structural modifications were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hypothesized alkylation was verified and structurally allocated to the OH-group of the C21 atom. The biological function of SM-alkylated GCs was investigated using GC-regulated dual-luciferase reporter gene assays and an ex vivo GC responsiveness assay coupled with real-time quantitative polymerase chain reaction (RT-qPCR). For the reporter gene assays, HEK293-cells were transiently transfected with a dual-luciferase reporter gene that is transcriptional regulated by a GC-response element. These cells were then incubated either with untreated or SM-derivatized HC, BM or DEX. Firefly-luciferase (Fluc) activity was determined 24 h after stimulation. Fluc-activity significantly decreased after stimulation with SM-pre-exposed GC dependent on the SM concentration. The ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to DEX revealed a transcriptional dysregulation of GC-regulated genes (FKBP5, IL1R2, and GILZ) after stimulation with SM-alkylated DEX. Our results present GCs as new biological targets of SM associated with a disturbance of hormone function.

Taste Receptors: New Players in Sperm Biology.

Taste receptors were first described as sensory receptors located on the tongue, where they are expressed in small clusters of specialized epithelial cells. However, more studies were published in recent years pointing to an expression of these proteins not only in the oral cavity but throughout the body and thus to a physiological role beyond the tongue. The recent observation that taste receptors and components of the coupled taste transduction cascade are also expressed during the different phases of spermatogenesis as well as in mature spermatozoa from mouse to humans and the overlap between the ligand spectrum of taste receptors with compounds in the male and female reproductive organs makes it reasonable to assume that sperm "taste" these different cues in their natural microenvironments. This assumption is assisted by the recent observations of a reproductive phenotype of different mouse lines carrying a targeted deletion of a taste receptor gene as well as the finding of a significant correlation between human male infertility and some polymorphisms in taste receptors genes. In this review, we depict recent findings on the role of taste receptors in male fertility, especially focusing on their possible involvement in mechanisms underlying spermatogenesis and post testicular sperm maturation. We also highlight the impact of genetic deletions of taste receptors, as well as their polymorphisms on male reproduction.

Immediate responses of the cockroach Blaptica dubia after the exposure to sulfur mustard.

The chemical agent sulfur mustard (SM) causes erythema, skin blisters, ulcerations, and delayed wound healing. It is accepted that the underlying molecular toxicology is based on DNA alkylation. With an expected delay, DNA damage causes impairment of protein biosynthesis and disturbance of cell division. However, using the cockroach model Blaptica dubia, the presented results show that alkylating compounds provoke immediate behavior responses along with fast changes in the electrical field potential (EFP) of neurons, suggesting that lesions of DNA are probably not the only effect of alkylating compounds. Blaptica dubia was challenged with SM or 2-chloroethyl-ethyl sulfide (CEES). Acute toxicity was objectified by a disability score. Physiological behavior responses (antennae pullback reflex, escape attempts, and grooming) were monitored after exposure. To estimate the impact of alkylating agents on neuronal activity, EFP recordings of the antennae and the thoracic ganglion were performed. After contact to neat SM, a pullback reflex of the antennae was the first observation. Subsequently, a striking escape behavior occured which was characterized by persistent movement of the legs. In addition, an instantaneous processing of the electrical firing pattern from the antennae to the descending ganglia was detectable. Remarkably, comparing the toxicity of the applied alkylating agents, effects induced by CEES were much more pronounced compared to SM. In summary, our findings document immediate effects of B. dubia after exposure to alkylating substances. These fast responses cannot be interpreted as a consequence of DNA alkylation. Therefore, the dogma that DNA alkylation is the exclusive cause for SM toxicity has to be questioned.

The IP3 R Binding Protein Released With Inositol 1,4,5-Trisphosphate Is Expressed in Rodent Reproductive Tissue and Spermatozoa.

Besides its capacity to inhibit the 1,4,5-trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co-localize with the proton pumping V-ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co-localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and/or the final fertilization process.

Inactivation of TRPM7 Kinase Targets AKT Signaling and Cyclooxygenase-2 Expression in Human CML Cells.

Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.

Morphine activates the E twenty six-like transcription factor-1/serum response factor pathway via extracellular signal-regulated kinases 1/2 in F11 cells derived from dorsal root ganglia neurons.

Morphine-induced signaling via opioid receptors (ORs) in dorsal root ganglia (DRG) neurons, the spinal cord, and various brain regions has been shown to modulate gene activity. Hitherto, little attention has been paid to extracellular signal-regulated kinases-1/2 (ERK-1/2)-mediated activation of the serum response factor (SRF) and ternary complex factors (TCFs) such as the E twenty six-like transcription factor-1 (ELK-1) in this context. Using TCF/SRF-dependent reporter gene constructs, a specific ERK-1/2 inhibitor and a dominant-negative ELK-1 mutant, we show herein that morphine activates ELK-1 via ERK-1/2 in DRG-derived F11 cells endogenously expressing µ and δ ORs. Previous studies with glioma cell lines such as NG108-15 cells attributed morphine-induced gene expression to the activation of the cAMP-responsive element binding protein (CREB). Thus, we also analyzed morphine-dependent activation of CREB in F11 and NG108-15 cells. In contrast to the CREB stimulation found in NG108-15 cells, we observed an inhibitory effect of morphine in F11 cells, indicating cell type-specific regulation of CREB by morphine. To obtain data about putative target genes of morphine-induced ELK-1/SRF activation, we analyzed mRNA levels of 15 ELK-1/SRF-dependent genes in cultured rat DRG neurons and F11 cells. We identified the early growth response protein-4 (EGR-4) as the strongest up-regulated gene in both cell types and observed ELK-1 activity-dependent activation of an EGR-4-driven reporter in F11 cells. Overall, we reveal an important role of ELK-1 for morphine-dependent gene induction in DRG-derived cells and propose that ELK-1 and EGR-4 contribute to the effects of morphine on neuronal plasticity.

Two-Pore Channels Regulate Inter-Organellar Ca2+ Homeostasis in Immune Cells.

Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca2+ from the endoplasmic reticulum as well as from acidic compartments, such as endolysosomes. For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca2+ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), were found to trigger the release of Ca2+ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca2+ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

CaMKIIalpha interacts with multi-PDZ domain protein MUPP1 in spermatozoa and prevents spontaneous acrosomal exocytosis.

The success of acrosomal exocytosis, a complex process with a variety of inter-related steps, relies on the coordinated interaction of participating signaling molecules. Since the acrosome reaction resembles Ca(2+)-regulated exocytosis in neurons, we investigated whether cognate neuronal binding partners of the multi-PDZ domain protein MUPP1, which recruits molecules that control the initial tethering and/or docking between the acrosomal vesicle and the plasma membrane, are also expressed in spermatozoa, and whether they contribute to the regulation of acrosomal secretion. We observed that CaMKIIalpha colocalizes with MUPP1 in the acrosomal region of epididymal spermatozoa where the kinase selectively binds to a region encompassing PDZ domains 10-11 of MUPP1. Furthermore, we found that pre-treating mouse spermatozoa with a CaMKII inhibitor that directly blocks the catalytic region of the kinase, as well as a competitive displacement of CaMKIIalpha from PDZ domains 10-11, led to a significant increase in spontaneous acrosomal exocytosis. Since Ca(2+)-calmodulin releases CaMKIIalpha from the PDZ scaffolding protein, MUPP1 represents a central signaling platform to dynamically regulate the assembly and disassembly of binding partners pertinent to acrosomal secretion, thereby precisely adjusting an increase in Ca(2+) to synchronized fusion pore formation.

Sensory neuron-specific MAS-related gene-X1 receptors resist agonist-promoted endocytosis.

Human sensory neuron-specific mas-related gene X1 receptors (hMrgX1s) belong to the superfamily of G protein-coupled receptors (GPCRs), bind cleavage products of pro-enkephalin with high affinity, and have been suggested to participate in pain sensation. Murine or rat MrgC receptors exhibit high similarities with hMrgX1 in terms of expression pattern, sequence homology, and binding profile. Therefore, rodents have been used as an in vivo model to explore the physiological functions and pharmacodynamics of the hMrgX1. Agonist-promoted receptor endocytosis significantly affects the pharmacodynamics of a GPCR but is not yet investigated for hMrgX1. Therefore, we analyzed the effects of prolonged agonist exposure on cell surface protein levels of hMrgX1 and murine or rat MrgC in human embryonic kidney 293, Cos, F11, and ND-C cells. We observed that hMrgX1 are resistant and both MrgC are prone to agonist-promoted receptor endocytosis. In Cos cells, coexpression of beta-arrestins strongly enhanced endocytosis of murine MrgC but did not alter cell surface expression of hMrgX1 receptors. These data define the hMrgX1 as one of the few members within the superfamily of GPCRs whose signaling is not regulated by agonist-promoted endocytosis and reveal species-specific differences in the regulation of Mrg receptor signaling. Given the importance of receptor endocytosis for the pharmacodynamics of a given ligand, our results may have a strong impact on the development of future drugs that suppose to control pain in humans but were tested in rodents.

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling.

During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptor-potential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naïve murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion.

Expression of Taste Receptor 2 Subtypes in Human Testis and Sperm.

Taste receptors (TASRs) are expressed not only in the oral cavity but also throughout the body, thus suggesting that they may play different roles in organ systems beyond the tongue. Recent studies showed the expression of several TASRs in mammalian testis and sperm, indicating an involvement of these receptors in male gametogenesis and fertility. This notion is supported by an impaired reproductive phenotype of mouse carrying targeted deletion of taste receptor genes, as well as by a significant correlation between human semen parameters and specific polymorphisms of taste receptor genes. To better understand the biological and thus clinical significance of these receptors for human reproduction, we analyzed the expression of several members of the TAS2Rs family of bitter receptors in human testis and in ejaculated sperm before and after in vitro selection and capacitation. Our results provide evidence for the expression of TAS2R genes, with TAS2R14 being the most expressed bitter receptor subtype in both testis tissue and sperm cells, respectively. In addition, it was observed that in vitro capacitation significantly affects both the expression and the subcellular localization of these receptors in isolated spermatozoa. Interestingly, α-gustducin and α-transducin, two Gα subunits expressed in taste buds on the tongue, are also expressed in human spermatozoa; moreover, a subcellular redistribution of both G protein α-subunits to different sub-compartments of sperm was registered upon in vitro capacitation. Finally, we shed light on the possible downstream transduction pathway initiated upon taste receptor activation in the male reproductive system. Performing ultrasensitive droplets digital PCR assays to quantify RNA copy numbers of a distinct gene, we found a significant correlation between the expression of TAS2Rs and TRPM5 (r = 0.87), the cation channel involved in bitter but also sweet and umami taste transduction in taste buds on the tongue. Even if further studies are needed to clarify the precise functional role of taste receptors for successful reproduction, the presented findings significantly extend our knowledge of the biological role of TAS2Rs for human male fertility.

The Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa.

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.

Role of Chemosensory TRP Channels in Lung Cancer.

Transient receptor potential (TRP) channels represent a large family of cation channels and many members of the TRP family have been shown to act as polymodal receptor molecules for irritative or potentially harmful substances. These chemosensory TRP channels have been extensively characterized in primary sensory and neuronal cells. However, in recent years the functional expression of these proteins in non-neuronal cells, e.g., in the epithelial lining of the respiratory tract has been confirmed. Notably, these proteins have also been described in a number of cancer types. As sensor molecules for noxious compounds, chemosensory TRP channels are involved in cell defense mechanisms and influence cell survival following exposure to toxic substances via the modulation of apoptotic signaling. Of note, a number of cytostatic drugs or drug metabolites can activate these TRP channels, which could affect the therapeutic efficacy of these cytostatics. Moreover, toxic inhalational substances with potential involvement in lung carcinogenesis are well established TRP activators. In this review, we present a synopsis of data on the expression of chemosensory TRP channels in lung cancer cells and describe TRP agonists and TRP-dependent signaling pathways with potential relevance to tumor biology. Furthermore, we discuss a possible role of TRP channels in the non-genomic, tumor-promoting effects of inhalational carcinogens such as cigarette smoke.

The multi PDZ domain protein MUPP1 as a putative scaffolding protein for organizing signaling complexes in the acrosome of mammalian spermatozoa.

Spermatozoa undergo complex sequences of precisely timed events during the process of fertilization. These priming events, which comprise capacitation, egg recognition, acrosome reaction, and sperm-oocyte fusion, are regulated by the activation of different intracellular signaling pathways. The efficacy and accuracy of signal transduction pathways often depend on the assembly of multiprotein signaling complexes, thereby coordinating and guiding the flow of regulatory information. To address the question whether PDZ-domain proteins, the most abundant protein interaction modules involved in the assembly of supramolecular signaling complexes, are present in rodent sperm, homologue of the RT-PCR approaches were performed with specific primer pairs for the vertebrate INAD-like PDZ domain protein MUPP1. The results revealed that this scaffolding protein, which comprises 13 different PDZ domains, is expressed in mouse testis. To obtain further support for the expression of the multi-PDZ domain protein MUPP1 in testicular tissue, immunohistochemical as well as immunocytochemical experiments were performed using a MUPP1-specific antibody. Detailed analyses of the spatial MUPP1-expression profile revealed that immunoreactivity is concentrated within the acrosomal region of round as well as elongated mouse spermatozoa. These results were confirmed in experimental approaches demonstrating that MUPP1 immunofluorescence was shed off from the acrosome region after acrosome reaction. To examine whether MUPP1 is also present in other mammalian sperm, immunocytochemical approaches were performed with isolated bovine as well as human sperm. The results revealed prominent MUPP1 expression which was restricted to the apical acrosomal region and, most notably, to the equatorial segment of the acrosome. The predominant expression profile of MUPP1 in sperm of different mammalian species suggests that this PDZ-domain protein may be involved in organizing signaling molecules mediating primary reactions of fertilization.

Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.

Selective G protein beta gamma-subunit compositions mediate phospholipase C activation in the vomeronasal organ.

Chemosensory neurons of the vomeronasal organ (VNO) are supposed to detect pheromones controlling social and reproductive behavior in most terrestrial vertebrates. Recent studies indicate that pheromone signaling in VNO neurons is mediated via phospholipase C (PLC) activation generating the two second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Since G alpha(i) and G alpha(o) predominantly expressed in VNO neurons are usually not involved in activating PLC, it was explored if PLC activation may be mediated by G beta gamma subunits. It was found that a scavenger for beta gamma dimers reduced the urine-induced IP3 formation in VNO preparations in a dose-dependent manner indicating a role for G beta gamma complexes. Towards an identification of the relevant G beta and G gamma subunit(s), PCR approaches as well as immunohistochemical experiments were performed. It was found that out of the five known G beta subtypes, only G beta2 was expressed in both G alpha(i) as well as G alpha(o) neurons. Experimental approaches focusing on the spatial expression profile of identified G gamma subtypes revealed that G gamma8-positive neurons are preferentially localized to the basal region of the vomeronasal epithelium, whereas G gamma2-reactive cells are restricted to the apical G alpha(i)-positive layer of the sensory epithelium. As IP3 formation induced upon stimulation with volatile urinary compounds was selectively blocked by G gamma2-specific antibodies whereas second messenger formation elicited upon stimulation with alpha2u globulin was inhibited by antibodies recognizing G gamma8, it is conceivable that PLC activation in the two populations of chemosensory VNO neurons is mediated by different G beta gamma complexes.