Localization of the angiopoietin receptors Tie-1 and Tie-2 on the primary cilia in the female reproductive organs

Blood vessel homeostasis and endothelial cell survival depend on proper signalling through angiopoietin receptors such as the receptor tyrosine kinases Tie-1 and Tie-2. We have studied the presence and subcellular localization of these receptors in murine female reproductive organs using confocal microscopy analysis of antibody stained tissue sections of ovary and oviduct. We show that Tie-2 principally localizes to primary cilia of the surface epithelium of the ovary, bursa and extra-ovarian rete ducts as well as to plasma membranes of ovarian theca and endothelial cells. Primary cilia of follicular granulosa cells were negative. Further, Tie-1 and Tie-2 localized to motile cilia of the oviduct. Western blotting detection and immunolocalization of anti-Tie-2 in ovary and oviduct were abolished by administration of an anti-Tie-2 blocking peptide, confirming antibody specificity. In a series of immunohistochemical analysis on human ovarian tissues we also observed a unique localization of Tie-2 to the primary cilia of ovarian surface epithelium. These observations are the first to show ciliary localization of angiopoietin receptors. Our results support the hypothesis that cilia of the female reproductive organs play a novel and important sensory role in relaying physiochemical changes from the extracellular environment to epithelial cells of the oviduct, the ovary and extra-ovarian tissues.     

A flagellar polycystin-2 homolog required for male fertility in Drosophila

A common inherited cause of renal failure, autosomal dominant polycystic kidney disease results from mutations in either of two genes, PKD1 and PKD2, which encode polycystin-1 and polycystin-2, respectively. Polycystin-2 has distant homology to TRP cation channels and associates directly with polycystin-1. The normal functions of polycystins are poorly understood, although recent studies indicate that they are concentrated in the primary cilia of a variety of cell types. In this report we identified a polycystin-2 homolog in Drosophila melanogaster; this homolog localized to the distal tip of the sperm flagella. A targeted mutation in this gene, almost there (amo), caused nearly complete male sterility. The amo males produced and transferred normal amounts of motile sperm to females, but mutant sperm failed to enter the female sperm storage organs, a prerequisite for fertilization. The finding that Amo functions in sperm flagella supports a common and evolutionarily conserved role for polycystin-2 proteins in both motile and nonmotile axonemal-containing structures.     

Structure and function of polycystin channels in primary cilia

Variants in genes which encode for polycystin-1 and polycystin-2 cause most forms of autosomal dominant polycystic disease (ADPKD). Despite our strong understanding of the genetic determinants of ADPKD, we do not understand the structural features which govern the function of polycystins at the molecular level, nor do we understand the impact of most disease-causing variants on the conformational state of these proteins. These questions have remained elusive because polycystins localize to several organelle membranes, including the primary cilia. Primary cilia are microtubule based organelles which function as cellular antennae. Polycystin-2 and related polycystin-2 L1 are members of the transient receptor potential (TRP) ion channel family, and form distinct ion channels in the primary cilia of disparate cell types which can be directly measured. Polycystin-1 has both ion channel and adhesion G-protein coupled receptor (GPCR) features—but its role in forming a channel complex or as a channel subunit chaperone is undetermined. Nonetheless, recent polycystin structural determination by cryo-EM has provided a molecular template to understand their biophysical regulation and the impact of disease-causing variants. We will review these advances and discuss hypotheses regarding the regulation of polycystin channel opening by their structural domains within the context of the primary cilia.     

Immunohistochemical localization of taurine in the rat ovary, oviduct, and uterus.

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133-1142, 2001)     

Direct interaction with Rab11a targets the epithelial Ca2+ channels TRPV5 and TRPV6 to the plasma membrane

TRPV5 and TRPV6 are the most Ca2+-selective members of the transient receptor potential (TRP) family of cation channels and play a pivotal role in the maintenance of Ca2+ balance in the body. However, little is known about the mechanisms controlling the plasma membrane abundance of these channels to regulate epithelial Ca2+ transport. In this study, we demonstrated the direct and specific interaction of GDP-bound Rab11a with TRPV5 and TRPV6. Rab11a colocalized with TRPV5 and TRPV6 in vesicular structures underlying the apical plasma membrane of Ca2+-transporting epithelial cells. This GTPase recognized a conserved stretch in the carboxyl terminus of TRPV5 that is essential for channel trafficking. Furthermore, coexpression of GDP-locked Rab11a with TRPV5 or TRPV6 resulted in significantly decreased Ca2+ uptake, caused by diminished channel cell surface expression. Together, our data demonstrated the important role of Rab11a in the trafficking of TRPV5 and TRPV6. Rab11a exerts this function in a novel fashion, since it operates via direct cargo interaction while in the GDP-bound configuration.     

Nitric oxide activates TRP channels by cysteine S-nitrosylation

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.     

Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been documented in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs are unknown in the female reproductive system of pigs. In this study, AQP1 immunoreactivity was detected in the capillary endothelium of the ovary. Distinct immunolabeling of capillary endothelium was also observed in the oviduct and uterus. AQP5 was expressed in flattened follicle cells of primordial follicles, granulosa cells of developing ovarian follicles, and muscle cells of the oviduct and uterus. Staining of AQP5 was also observed in the epithelial cells of the oviduct and uterine epithelium. AQP9 immunoreactivity was observed in granulosa cells of developing follicles. AQP9 was also localized in the luminal epithelial cells of the oviduct and uterine epithelia cells. This is, to our knowledge, the first study that shows tissue expression and cellular and subcellular localization of AQPs in the reproductive system of the female pig. Moreover, these results suggest that several subtypes of the AQPs (AQP1, 5, and 9) are involved in regulation of water homeostasis in the reproductive system of gilts.     

Hormone-sensitive lipase expression and IHC localization in the rat ovary, oviduct, and uterus

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.     

Polycystins: what polycystic kidney disease tells us about sperm

Experimental evidence indicates that the membrane-associated proteins polycystin-1 and polycystin-2 operate as a receptor-calcium channel complex that regulates signaling pathways essential for modulation of renal tubulogenesis. Polycystic kidney disease is characterized by defective renal tubular structure and results from mutations in either PKD1 or PKD2 genes. Recent data suggest that polycystin-1 and polycystin-2 might localize to primary cilium in principal cells of renal collecting tubules and are thought to act as mechanosensors of fluid flow and contents. Ciliary bending by fluid flow or mechanical stimulation induce Ca(2+) release from intracellular stores, presumably to modulate ion influx in response to tubular fluid flow. Polycystins are also emerging as playing a significant role in sperm development and function. Drosophila polycystin-2 is associated with the head and tail of mature sperm. Targeted disruption of the PKD2 homolog results in nearly complete male sterility without disrupting spermatogenesis. Mutant sperm are motile but are unable to reach the female storage organs (seminal receptacles and spermathecae). The sea urchin polycystin-1-equivalent suPC2 colocalizes with the polycystin-1 homolog REJ3 to the plasma membrane over the acrosomal vesicle. This localization site suggests that the suPC2-REJ3 complex may function as a cation channel mediating acrosome reaction when sperm contact the jelly layer surrounding the egg at fertilization. Future studies leading to the identification of specific ligands for polycystins, including the signaling pathways, might define the puzzling relationship between renal tubular morphogenesis and sperm development and function.     

TRP channels: an overview

The TRP ("transient receptor potential") family of ion channels now comprises more than 30 cation channels, most of which are permeable for Ca2+, and some also for Mg2+. On the basis of sequence homology, the TRP family can be divided in seven main subfamilies: the TRPC ('Canonical') family, the TRPV ('Vanilloid') family, the TRPM ('Melastatin') family, the TRPP ('Polycystin') family, the TRPML ('Mucolipin') family, the TRPA ('Ankyrin') family, and the TRPN ('NOMPC') family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data on the roles of TRPs in a variety of tissues and species, including mammals, insects, and yeast. The present review summarizes the most pertinent recent evidence regarding the structural and functional properties of TRP channels, focusing on the regulation and physiology of mammalian TRPs.     

STAM and Hrs down-regulate ciliary TRP receptors

Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.     

dTULP,the Drosophila melanogaster Homolog of Tubby,Regulates Transient Receptor Potential Channel Localization in Cilia

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.     

Casein kinase II and calcineurin modulate TRPP function and ciliary localization

Cilia serve as sensory devices in a diversity of organisms and their defects contribute to many human diseases. In primary cilia of kidney cells, the transient receptor potential polycystin (TRPP) channels polycystin-1 (PC-1) and polycystin-2 (PC-2) act as a mechanosensitive channel, with defects resulting in autosomal dominant polycystic kidney disease. In sensory cilia of Caenorhabditis elegans male-specific neurons, the TRPPs LOV-1 and PKD-2 are required for mating behavior. The mechanisms regulating TRPP ciliary localization and function are largely unknown. We identified the regulatory subunit of the serine-threonine casein kinase II (CK2) as a binding partner of LOV-1 and human PC-1. CK2 and the calcineurin phosphatase TAX-6 modulate male mating behavior and PKD-2 ciliary localization. The phospho-defective mutant PKD-2(S534A) localizes to cilia, whereas a phospho-mimetic PKD-2(S534D) mutant is largely absent from cilia. Calcineurin is required for PKD-2 ciliary localization, but is not essential for ciliary gene expression, ciliogenesis, or localization of cilium structural components. This unanticipated function of calcineurin may be important for regulating ciliary protein localization. A dynamic phosphorylation-dephosphorylation cycle may represent a mechanism for modulating TRPP activity, cellular sensation, and ciliary protein localization.     

Role and regulation of TRP channels in neutrophil granulocytes

Members of the transient receptor potential (TRP) family for which mRNA can be demonstrated in neutrophil granulocytes with RT-PCR include TRPC6 (as only "short" TRP), TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6. When these are analyzed in heterologous overexpression experiments, TRPM2 is the only cation channel with characteristic properties that can be used as fingerprint to provide functional evidence for its expression in neutrophil granulocytes. As cells transfected with TRPM2, neutrophil granulocytes display non-selective cation currents and typical channel activity evoked by intracellular ADP-ribose and NAD. Thus, stimulation of TRPM2 is likely to occur after activation of CD38 (producing ADP-ribose) and during the oxidative burst (enhancing the NAD concentration). This novel mode of cation entry regulation may be of particular importance for the response of granulocytes to chemoattractants. TRPV6 is a likely but not exclusive candidate as subunit of the channels mediating store-operated Ca2+ entry (SOCE). Evidence for SOCE in granulocytes has been presented with the fura-2 technique but not with electrophysiological methods although Ca2+-selective store-operated currents can be demonstrated in HL-60 cells, a cell culture model of neutrophil granulocytes.     

Calcium signaling and polycystin-2

Polycystic kidney disease (PKD) is caused by mutations in two genes, PKD1 and PKD2, which encode for the proteins, polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Although disease-associated mutations have been identified in these two proteins, the sequence of molecular events leading up to clinical symptoms is still unknown. PC1 resides in the plasma membrane and it is thought to function in cell-cell and cell-matrix interactions, whereas PC2 is a calcium (Ca2+) permeable cation channel concentrated in the endoplasmic reticulum. Both proteins localize to the primary cilia where they function as a mechanosensitive receptor complex allowing the entry of Ca2+ into the cell. The downstream signaling pathway involves activation of intracellular Ca2+ release channels, especially the ryanodine receptor (RyR), but subsequent steps are still to be identified. Elucidation of the signaling pathway involved in normal PC1/PC2 function, the functional consequences of PC1/PC2 mutation, and the role of Ca2+ signaling will all help to unravel the molecular mechanisms of cystogenesis in PKD.     

Expression and distribution of three transient receptor potential vanilloid(TRPV) channel proteins in human odontoblast-like cells

Odontoblasts have been suggested to contribute to nociceptive sensation in the tooth via expression of the transient receptor potential (TRP) channels. The TRP channels as a family of nonselective cation permeable channels play an important role in sensory transduction of human. In this study, we examined the expression of transient receptor potential vanilloid-1 (TRPV1), transient receptor potential vanilloid-2 (TRPV2) and transient receptor potential vanilloid-3 (TRPV3) channels in native human odontoblasts (HODs) and long-term cultured human dental pulp cells with odontoblast phenotyoe (LHOPs) obtained from healthy wisdom teeth with the use of immunohistochemistry (IHC), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR),western blotting (WB) and immunoelectron microscopy (IEM) assay. LHOPs samples were made into ultrathin sections, mounted on nickel grids, floated of three TRPV antibodies conjugated with 10 nm colloidal gold particles and observed under IEM at 60,000 magnifications. The relative intracellular distributions of these three channels were analyzed quantitatively on IEM images using a robust sampling, stereological estimation and statistical evaluation method. The results of IHC and IF convinced that TRPV1, TRPV2 and TRPV3 channels were expressed in native HODs and (LHOPs). The result of qRT-PCR and WB confirmed that the gene and protein expression of TRPV1, TRPV2, and TRPV3 channels and TRPV1 mRNA are more abundantly expressed than TRPV2 and TRPV3 in HODs (P?<?0.05). Quantitative analysis of IEM images showed that the relative intracellular distributions of these three channels are similar, and TRPV1, TRPV2 and TRPV3 proteins were preferential labeled in human odontoblast processes, mitochondria, and endoplasmic reticulum. Thus, HODs could play an important role in mediating pulp thermo-sensation due to the expression of these three TRPV channels. The difference of relative intracellular distributions of three channels suggests that special structures such as processes may have an important role to sensing of the outer stimuli first.     

Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca(2+) transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5-20 μM) -induced Ca(2+) transients were blocked by capsazepine (CPZ) (10 μM), the TRPV4 activator 4α-PDD (10 μM) -induced Ca(2+) increases were reduced by ruthenium-red (RuR) (20 μM). Different heating (<40°C or >43°C) led to Ca(2+) increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca(2+) transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.     

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.