Basolateral Stimulation Research Articles

A22 USING INTESTINAL ORGANOIDS TO CHARACTERIZE THE NAIP-NLRC4 INFLAMMASOME RESPONSE IN THE INTESTINAL EPITHELIUM

Abstract Background The NAIP-NLRC4 inflammasome is an important innate defence mechanism in intestinal epithelial cells (IECs) that protects the gut from invasive pathogens. NAIP-NLRC4 activation triggers pyroptotic cell death, releases active interleukin (IL)-18, and promotes the expulsion of infected enterocytes. Dysregulated inflammasomes result in exaggerated inflammation of the intestinal mucosa which is characterisitic of inflammatory bowel disease (IBD). There is increasing evidence that inflammasomes in IEC behave differently than in immune cells. However, a majority of inflammasome pathway characterization is studied immune cells. In addition, NAIP-NLRC4 activity in macrophages has been shown to be important for regulating the expression of nitric oxide (Nos2). The potential transcriptomic impact of NAIP-NLRC4 activity in IECs has yet to be explored. Purpose We will use intestinal orgnaoids to systematically characterize the NAIP-NLRC4 inflammasome pathway and identify the transcriptomic impact of NAIP-NLRC4 activity in IECs. To better define the role of NAIP-NLRC4 activity during a physiological infection, we have developed a novel ex vivo model of Shigella infection in 3D organoids. Method Organoids were derived from the ileal crypts of wild type (WT) and Nlrc4-/-, Casp1-/-, Pycard-/-, or Tlr5-/- mice and stimulated with Pam3CSk4, LPS, or flagellin. Inflammasome activation was assessed by Western blot (WB) and propidium iodide uptake. WT and Nlrc4-/- organoids were infected with WT or a non-invasive Shigella mutant and the inflammasome response was evaluated by WB and a colony forming unit assay. WT and Nlrc4-/- organoids were stimulated with flagellin and gene expression was assessed by RT-qPCR. Result(s) Basolateral organoid stimulation with bacterial ligands revealed a novel response of IECs to bacterial flagellin that results in pyroptosis and IL-18 processing. Basolateral internalization of flagellin occurred in a TLR5-independent manner. Inflammasome activation by flagellin was fully abrogated in Nlrc4-/- and Casp1-/- organoids while only IL-18 processing was affected in Pycard-/- organoids. Infection with only WT Shigella induced inflammasome activation in an NLRC4-dependent manner. Interestingly, flagellin stimulation of WT but not Nlrc4-/- organoids led to increased expression of Nos2. Furthermore, Nlrc4-/- organoids had significantly lower expression levels of cytokine genes Ccl20, Cxcl1, and Tnf following inflammasome activation. Conclusion(s) Our study demonstrates an integral role for epithelial NAIP-NLRC4 inflammasomes in the response to bacterial flagellin and Shigella infection. We have uncovered a novel response of IECs to basolateral flagellin stimulation and revealed that NAIP-NLRC4 is important for the transcriptional regulation of inflammatory genes. Further work will examine how NAIP-NLRC4 activation controls inflammation and epithelial integrity by analyzing the NLRC4-dependent transcriptome and the effects on cell proliferation, differentiation, and barrier integrity. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

Quantitative Assessment of the Apical and Basolateral Membrane Expression of VEGFR2 and NRP2 in VEGF-A-stimulated Cultured Human Umbilical Vein Endothelial Cells.

Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood-retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.

A55 INVESTIGATING THE ROLE OF ENDOGENOUS INTERLEUKIN-10 IN INTESTINAL EPITHELIAL CELLS AND HOMEOSTASIS

Abstract Background IL-10 is appreciated for its potent anti-inflammatory effects on leukocytes in mucosal immunity. However, far less attention has been paid to the impact of IL-10 on epithelial cells, which make up the crucial barrier interface between the host mucosa and the external environment. Furthermore, most studies examine the effects of exogenous IL-10, disregarding the possible presence and function of autocrine or paracrine IL-10 in the epithelium. Aims Using ex vivo organoids we aimed to examine the small intestinal epithelium for IL-10 and dissect any role for endogenously produced cytokine. Methods We growed small intestinal organoids (enteroids) from crypts isolated from C57BL/6 mice (WT) and IL-10-gene knockout mice (IL-10KO). Cellular markers were characterized through qpCR, while IL-10 and IL-10 receptor localization was characterized though immunofluorescence. Results We discovered that cells in WT enteroids expressed IL-10 and IL-10R1 constitutively throughout development. Immunofluorescent staining revealed that IL-10 localizes to Paneth cells and appears to be secreted apically. Having established that IL-10 is secreted in enteroids, we compared enteroids from IL-10KO versus WT mice. IL-10KO enteroids developed to morphologically resemble WT enteroids; however, we detected an imbalance with lower secretory cell markers over absorptive cell types in the IL-10KO enteroids, measured as less mRNA for lysozyme, cryptdins and mucin-2. Addition of IL-10 to IL-10KO enteroids did not correct these defects, but did ameliorate the lineage balance by reducing absorptive cell lineage markers (sucrose isomaltase). IL-10R1 was localized on both apical and basolateral side of cell in enteroids. We suspect that epithelial-derived IL-10 likely acts on apical IL-10R, which may conduct a different response from basolateral receptor stimulation. Conclusions In conclusion, IL-10 is present in the small intestinal epithelium; more remains to be determined regarding the role this cytokine plays in gut development and homeostasis. Funding Agencies NSERC

Basolateral activation with TLR agonists induces polarized cytokine release and reduces barrier function in RPE in vitro

PurposeSystemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability.MethodsRPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining.ResultsNone of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells.ConclusionsDanger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

Fully synthetic matrices for in vitro culture of primary human intestinal enteroids and endometrial organoids

Epithelial organoids derived from human donor tissues are important tools in fields ranging from regenerative medicine to drug discovery. Organoid culture requires expansion of stem/progenitor cells in Matrigel, a tumor-derived extracellular matrix (ECM). An alternative completely synthetic ECM could improve reproducibility, clarify mechanistic phenomena, and enable human implantation of organoids. We designed synthetic ECMs with tunable biomolecular and biophysical properties to identify gel compositions supporting human tissue-derived stem/progenitor epithelial cells as enteroids and organoids starting with single cells rather than tissue fragments. The synthetic ECMs consist of 8-arm PEG-macromers modified with ECM-binding peptides and different combinations of integrin-binding peptides, crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation, and tuned to exhibit a range of biophysical properties. A gel containing an α2β1 integrin-binding peptide (GFOGER) and matrix binder peptides grafted to a 20 kDa 8-arm PEG macromer showed the most robust support of human duodenal and colon enteroids and endometrial organoids. In this synthetic ECM, human intestinal enteroids and endometrial organoids emerge from single cells and show cell-specific and apicobasal polarity markers upon differentiation. Intestinal enteroids, in addition, retain their proliferative capacity, are functionally responsive to basolateral stimulation, express canonical markers of intestinal crypt cells including Paneth cells, and can be serially passaged. The success of this synthetic ECM in supporting human postnatal organoid culture from multiple different donors and from both the intestine and endometrium suggests it may be broadly useful for other epithelial organoid culture.

Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI-H520 squamous cells increased intracellular calcium levels and granulocyte macrophage-colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air-liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

Inhibitory Control of Basolateral Amygdalar Transmission to the Prefrontal Cortex by Local Corticotrophin Type 2 Receptor.

BackgroundBasolateral amygdalar projections to the prefrontal cortex play a key role in modulating behavioral responses to stress stimuli. Among the different neuromodulators known to impact basolateral amygdalar-prefrontal cortex transmission, the corticotrophin releasing factor (CRF) is of particular interest because of its role in modulating anxiety and stress-associated behaviors. While CRF type 1 receptor (CRFR1) has been involved in prefrontal cortex functioning, the participation of CRF type 2 receptor (CRFR2) in basolateral amygdalar-prefrontal cortex synaptic transmission remains unclear.MethodsImmunofluorescence anatomical studies using rat prefrontal cortex synaptosomes devoid of postsynaptic elements were performed in rats with intra basolateral amygdalar injection of biotinylated dextran amine. In vivo microdialysis and local field potential recordings were used to measure glutamate extracellular levels and changes in long-term potentiation in prefrontal cortex induced by basolateral amygdalar stimulation in the absence or presence of CRF receptor antagonists.ResultsWe found evidence for the presynaptic expression of CRFR2 protein and mRNA in prefrontal cortex synaptic terminals originated from basolateral amygdalar. By means of microdialysis and electrophysiological recordings in combination with an intra-prefrontal cortex infusion of the CRFR2 antagonist antisauvagine-30, we were able to determine that CRFR2 is functionally positioned to limit the strength of basolateral amygdalar transmission to the prefrontal cortex through presynaptic inhibition of glutamate release.ConclusionsOur study shows for the first time to our knowledge that CRFR2 is expressed in basolateral amygdalar afferents projecting to the prefrontal cortex and exerts an inhibitory control of prefrontal cortex responses to basolateral amygdalar inputs. Thus, changes in CRFR2 signaling are likely to disrupt the functional connectivity of the basolateral amygdalar-prefrontal cortex pathway and associated behavioral responses.

Stem Cells are the Principal Intestinal Epithelial Responders to Bacterial Antigens

Intestinal epithelial cells are capable of mounting adequate antimicrobial inflammatory responses to pathogens, as well as tolerogenic responses to commensals. We studied whether epithelial responsiveness depends on the surface where stimulation takes place, i.e. basolateral vs. apical, the state of cell differentiation and the location along the intestinal longitudinal axis. Organoids grown from different intestinal locales are equally responsive to microbes. However, we predominantly found epithelial responses upon basolateral stimulation and major differences when we determined specific responses of various epithelial cells along the crypt-villus axis. Specifically, we identified intestinal stem cells as the major responder, whereas epithelial differentiation to enterocytes was associated with attenuated responsiveness. We establish that the decrease in NFKB-dependent responses in differentiated cells is mediated by a post-transcriptional decrease in RNA stability. These findings provide new insights into the complexity of epithelial responses to microbes and identify post-transcriptional processes as a key controller of non-responsiveness.

Compartmentalized Response of IL-6/STAT3 Signaling in the Colonic Mucosa Mediates Colitis Development.

A single layer of polarized epithelial cells lining the colonic mucosa create a semipermeable barrier indispensable for gut homeostasis. The role of intestinal epithelial cell (IEC) polarization in the maintenance of the epithelial homeostasis and in the development of inflammatory bowel diseases is not fully understood. In this review, now we report that IEC polarization plays an essential role in the regulation of IL-6/STAT3 signaling in the colonic mucosa. Our results demonstrate that autocrine STAT3 activation in IECs is mediated by the apical secretion of IL-6 in response to the basolateral stimulation with IFN-γ. This process relies on the presence of functional, IFN-γ-producing CD4+ T cells. In the absence of basolateral IFN-γ, the compartmentalization of the IL-6/STAT3 signaling is disrupted, and STAT3 is activated mainly in macrophages. Thus, in this study, we show that during inflammation, IFN-γ regulates IL-6/STAT3 signaling in IEC in the colonic mucosa.

Non-canonical Inflammasome-Mediated IL-1β Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent.

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.

The combination of endolysosomal escape and basolateral stimulation to overcome the difficulties of "easy uptake hard transcytosis" of ligand-modified nanoparticles in oral drug delivery.

Ligand-modified nanoparticles (NPs) are an effective tool to increase the endocytosis efficiency of drugs, but these functionalized NPs face the drawback of "easy uptake hard transcytosis" in the oral delivery of proteins and peptides. Adversely, the resulting deficiency in transcytosis has not attracted much attention. Herein, NPs modified with the low-density lipoprotein receptor (LDLR) ligand NH2-C6-[cMPRLRGC]c-NH2, i.e., peptide-22 (P22NPs) were fabricated to investigate strategies related to the enhancement of transcytosis. By systematically studying the intracellular trafficking of NPs, it was found that reduced transcytosis might be associated with the entrapment of P22NPs in endosomes or lysosomes and limited basolateral exocytosis. On this basis, the prevention of the endolysosomal entrapment of NPs and the acceleration of basolateral exocytosis should be considered as strategies to enhance the transcytosis of NPs. By screening chemicals that could help the endosomal/lysosomal escape of chemicals related to LDLR-mediated transcytosis, it was shown that hemagglutinin-2 (HA2) and metformin had higher abilities to enhance the exocytosis of P22NPs. The transcytosis efficiencies of insulin loaded in P22NPs were also investigated, and a 3.2-fold increase in transcytosis was observed in comparison with free insulin. The transcytosis efficiencies of insulin could be further increased by the addition of metformin or HA2 (3.6-fold or 4.1-fold higher than that of free insulin). Inspiringly, the simultaneous addition of the abovementioned two chemicals led to the highest transcytosis efficiency of insulin, which was up to 5.1-fold higher than that of free insulin. These results demonstrated that endolysosomal entrapment and basolateral exocytosis are two of the most important limiting steps for the "easy uptake hard transcytosis" of orally administered ligand-modified NPs. Moreover, our work provides a new point of view for the design of novel oral drug delivery systems.

Polarized secreted IL-17C is a mediator of respiratory epithelial innate immune response

The IL-17 family of cytokines consists of at least six members (IL-17A to -F). IL-17 directly activates epithelial cells leading to the expression of inflammatory mediators and antimicrobial factors. In this study, our aim was to examine the expression and function of IL-17C in respiratory epithelial cells during bacterial infection. In vitro studies showed that common bacterial pathogens, such as Pseudomonas aeruginosa and Haemophilus influenzae, and ligands of Toll-like receptors 3 and 5 (flagellin, polyI:C) induced the expression and release of IL-17C in cultured human bronchial epithelial cells. Differentiated bronchial epithelial cells secreted IL-17C into the basolateral compartment and the secretion of IL-17C was increased in response to basolateral stimulation as compared to apical stimulation with bacterial stimuli. Pharmacological inhibition of intracellular calcium release and calcium chelators inhibited the secretion of IL-17C whereas the release of the inflammatory mediator IL-6 was not affected. IL-17C enhanced inflammatory responses of respiratory epithelial cells infected with P. aeruginosa. These data show that calcium-dependent signaling cascades mediate the polarized secretion of IL-17C and that IL-17C mediates innate immune responses of respiratory epithelial cells.

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Transformation of polarized epithelial cells by apical mistrafficking of epiregulin

Establishment and maintenance of apico-basolateral trafficking pathways are critical to epithelial homeostasis. Loss of polarity and trafficking fidelity are thought to occur as a consequence of transformation; however, here we report that selective mistrafficking of the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) from the basolateral to the apical cell surface drives transformation. Normally, EREG is preferentially delivered to the basolateral surface of polarized Madin-Darby canine kidney cells. EREG basolateral trafficking is regulated by a conserved tyrosine-based basolateral sorting motif in its cytoplasmic domain (YXXΦ: Y(156)ERV). Both Y156 and V159 are required for basolateral sorting of EREG, because Y156A and V159G substitutions redirect EREG to the apical cell surface. We also show that basolateral sorting of EREG is adaptor protein 1B-independent. Apical mistrafficking of EREG has a distinctive phenotype. In contrast to transient EGFR tyrosine phosphorylation after basolateral EREG stimulation, apical EREG leads to prolonged EGFR tyrosine phosphorylation, which may be related, at least in part, to a lack of negative regulatory Y1045 phosphorylation and subsequent ubiquitylation. Notably, Madin-Darby canine kidney cells stably expressing apically mistrafficked EREG form significantly larger, hyperproliferative, poorly differentiated, and locally invasive tumors in nude mice compared with WT EREG-expressing cells.

Basolateral BMP Signaling in Polarized Epithelial Cells

Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-β or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

P2Y 2 receptor stimulation increases K secretion by activating K Ca 3.1 potassium channels in human mammary epithelial cells

Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP (10 μM) or ATP-γ-S (10 μM) stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (Isc), consistent with activation of an apical K conductance. In contrast, basolateral stimulation with UTP (10 μM) produced a longer duration increase in Isc that was blocked by 5 μM benzamil. Inhibition of Ca2+ mobilization using BAPTA-AM abolished these changes in Isc. Moreover, apical pretreatment with charybdotoxin (200 nM), blocked the effect on Isc by >80% and a similar degree of inhibition was observed with clotrimazole (10 μM) and TRAM-34 (0.5 μM). Silencing expression of KCa3.1 by transfection of HME cells with an shRNA containing lentivirus construct targeting the channel produced ~80% inhibition of mRNA expression and 71% inhibition of the apical Isc response. In addition, silencing the P2Y2 receptor by expression of an shRNA containing lentivirus construct reduced the level of P2Y2 mRNA by 75% and inhibited the apical effects of 10 μM ATP-γ-S by 65%. These results suggest that P2Y2 receptors mediate the effects of purinoceptor agonists on K secretion by regulating the activity of KCa3.1 channels expressed in the apical membrane of HME cells.

Establishment of Cell-Cell Cross Talk in the Epididymis: Control of Luminal Acidification

Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte. Spermatozoa acquire these crucial functions in the epididymis. A low luminal bicarbonate (HCO(3)(-)) concentration and low pH keep sperm quiescent during their maturation and storage in this organ. This review describes how epididymal epithelial cells work in a concerted manner, together with spermatozoa, to establish and maintain this acidic luminal environment. Clear cells express the proton-pumping ATPase (V-ATPase) in their apical membrane and actively secrete protons. HCO(3)(-) induces V-ATPase accumulation in apical microvilli in clear cells via HCO(3)(-)-sensitive adenylyl cyclase-dependent cAMP production. HCO(3)(-) is secreted from principal cells following basolateral stimulation, to transiently "prime" spermatozoa before ejaculation. Luminal ATP and adenosine also induce V-ATPase apical accumulation in clear cells via activation of P2 and P1 receptors, respectively. ATP is released into the lumen from sperm and principal cells and is then metabolized into adenosine by local nucleotidases. In addition, the V-ATPase is regulated by luminal angiotensin II via activation of basal cells, which can extend narrow body projections that cross the tight junction barrier. Basal cells then secrete nitric oxide, which diffuses out to stimulate proton secretion in clear cells via activation of the cGMP pathway. Thus, an elaborate communication network is present between principal cells and clear cells, and between basal cells and clear cells, to control luminal acidification. Monitoring and decoding these "intercellular conversations" will help define pathophysiologic mechanisms underlying male infertility.

Localized cortical injections of ethosuximide suppress spike-and-wave activity and reduce the resistance to kindling in genetic absence epilepsy rats (GAERS)

Models of genetic absence epilepsy are resistant to secondary generalization of focal limbic seizures. This correlates with the postnatal development of spike-and-wave discharges (SWDs), a hallmark of absence seizures arising from a cortical focus in the perioral region of somatosensory cortex. Ethosuximide injected at this site suppresses SWDs. The effect of this suppression on kindling in "Genetic Absence Epilepsy Rats from Strasbourg" (GAERS), has been compared for postnatal 30 day (PN30) rats having immature SWDs and adult (>4 months) rats having mature SWDs. Non-epileptic Wistar and GAERS rats were implanted with a basolateral amygdaloid stimulation electrode, bilateral injection cannulas into the cortical perioral focus, and cortical recording electrodes. Following recovery cortical injections of ethosuximide or saline were made and after 30min rats were given 36 stimulations or until Racine's stage 5 seizures were produced. All Wistar rats (PN30 and adult) treated with saline or ethosuximide reached stage 5. Of GAERS given saline, 33% (PN30) and 43% (adults) were resistant to kindling; after ethosuximide pups behaved like Wistars, but adults showed a delay in kindling relative to Wistars. These findings imply that mechanisms underlying kindling resistance are related but not limited to SWD activity in animals with genetic absence epilepsy.

Enhanced ENaC and KCa3.1 expression by hydrocortisone increases Na+ absorption in human mammary epithelial cells.

Immortalized human mammary epithelial cells express apical ENaC sodium channels and basolateral KCa3.1 potassium channels. Treatment of monolayers with hydrocortisone (0.5 μg/ml) for 48 hours enhanced mRNA expression for α, β and γ ENaC subunits by 7.3, 10.5 and 9.6 fold as determined by quantitative RT‐PCR. Additionally, KCa3.1 mRNA expression was enhanced by hydrocortisone by 8.1 fold, however no increase in mRNA expression was detected for the BK (Slo1) potassium channel, which had a higher basal level of expression than KCa3.1 (Slo1 CT = 23.8; KCa3.1 CT = 27.7). Hydrocortisone also increased expression of the P2Y6 receptor by 5.5 fold without significantly altering P2Y1, P2Y2 or P2Y4 receptor expression. Basolateral stimulation of hydrocortisone pretreated monolayers with UTP (10 μM) resulted in a significantly greater increase in short circuit current (Isc) compared to untreated control monolayers (peak ΔIsc + hydrocortisone = 20 ± 1 μA, control = 9.5 ± 2 μA). In addition, the duration of the Isc response was significantly lower in the absence of hydrocortisone with less oscillation. These results indicate that KCa3.1 and P2Y6 receptor expression are up‐regulated in parallel with ENaC channels in the presence of hydrocortisone resulting in enhanced electrogenic Na+ absorption following basolateral stimulation with UTP.