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Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells.

ABSTRACT: Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.

SUBMITTER: Nepal N

PROVIDER: S-EPMC8303499 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mechanism of Na-K-ATPase Inhibition by PGE2 in Intestinal Epithelial Cells.

Project description:The primary means of intestinal absorption of nutrients by villus cells is via Na-dependent nutrient co-transporters located in the brush border membrane (BBM). These secondary active co-transport processes require a favorable transcellular Na gradient that is provided by Na-K-ATPase. In chronic enteritis, malabsorption of essential nutrients is partially due to inhibition of villus Na-K-ATPase activity mediated by specific immune inflammatory mediators that are known to be elevated in the inflamed mucosa. However, how Prostaglandin E2 (PGE2), a specific mediator of nutrient malabsorption in the villus BBM, may mediate the inhibition of Na-K-ATPase is not known. Therefore, this study aimed to determine the effect of PGE2 on Na-K-ATPase in villus cells and define its mechanism of action. In vitro, in IEC-18 cells, PGE2 treatment significantly reduced Na-K-ATPase activity, accompanied by a significant increase in the intracellular levels of cyclic Adenosine Monophosphate (cAMP). The treatment with cAMP analog 8-Bromo-cAMP mimicked the PGE2-mediated effect on Na-K-ATPase activity, while Rp-cAMP (PKA inhibitor) pretreatment reversed the same. The mechanism of inhibition of PGE2 was secondary to a transcriptional reduction in the Na-K-ATPase α1 and β1 subunit genes, which was reversed by the Rp-cAMP pretreatment. Thus, the PGE2-mediated activation of the PKA pathway mediates the transcriptional inhibition of Na-K-ATPase activity in vitro.

| S-EPMC8066871 | biostudies-literature

Leukotriene D4 induces association of active RhoA with phospholipase C-gamma1 in intestinal epithelial cells.

Project description:It has been previously suggested that leukotriene-induced Ca2+ signalling is mediated through a Rho-dependent process, but neither direct activation of Rho nor a mechanism underlying such signalling has been reported. Accordingly, we used the Rhotekin binding assay to assess RhoA activation in intestinal epithelial cells and observed that RhoA was activated by leukotriene D4 (LTD4). We also found that, within 15 s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein betagamma (Gbetagamma) and phospholipase C-gamma1 (PLC-gamma1) in the plasma membrane, as evidenced by the results of co-immunoprecipitation, glutathione S-transferase (GST) pulldown assays, and confocal microscopy. Amounts of RhoA increased in both Gbeta and PLC-gamma1 immunoprecipitates within 15 s of LTD4 treatment. An interaction between RhoA, Gbetagamma and PLC-gamma1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gbetagamma and PLC-gamma1 from cell lysates in an agonist-dependent manner. Such an association is also substantiated by our confocal immunofluorescence results, which revealed that LTD4 induction increased co-localization of constitutively active RhoA and PLC-gamma1 to the plasma membrane of cells transfected with enhanced green fluorescent protein L63RhoA. Furthermore, microinjection of neutralizing RhoA antibodies, but not control antibodies, significantly reduced LTD4-induced Ca2+ mobilization. Our results are the first to demonstrate a LTD4-induced activation of RhoA and more importantly its association with PLC-gamma1, which are essential for the PLC-gamma1-mediated calcium mobilization.

| S-EPMC1222665 | biostudies-other

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

Project description:Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by ?-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1?, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

| S-EPMC5003151 | biostudies-literature

Leukotriene D4-triggered Transcriptome

Project description:Human umbilical vein endothelial cells (HUVEC) or the human macrophage cell line, Mono-Mac-6, treated with Leukotriene D4 for 1 hour Keywords = Leukotriene-Transcriptome Keywords = Mono-Mac-6 Keywords = HUVEC Keywords: other

2004-08-20 | GSE1644 | GEO

A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.

Project description:FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na+ and K+ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.

| S-EPMC2642742 | biostudies-literature

Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes.

Project description:BACKGROUND:Leukotriene B(4) (LTB(4)) and cysteinyl leukotrienes (cysLTs) are important immune mediators, often found concomitantly at sites of inflammation. Although some of the leukotriene-mediated actions are distinctive (e.g., bronchial constriction for cysLTs), many activities such as leukocyte recruitment to tissues and amplification of inflammatory responses are shared by both classes of leukotrienes. OBJECTIVE:We used human monocytes to characterize leukotriene-specific signaling, gene expression signatures, and functions and to identify interactions between LTB(4)- and cysLTs-induced pathways. METHODS:Responsiveness to leukotrienes was assessed using oligonucleotide microarrays, real-time PCR, calcium mobilization, kinase activation, and chemotaxis assays. RESULTS:Human monocytes were found to express mRNA for high- and low-affinity LTB(4) receptors, BLT(1) and BLT(2), but signal predominantly through BLT(1) in response to LTB(4) stimulation as shown using selective agonists, inhibitors, and gene knock down experiments. LTB(4) acting through BLT(1) coupled to G-protein ? inhibitory subunit activated calcium signaling, p44/42 mitogen-activated protein kinase, gene expression, and chemotaxis. Twenty-seven genes, including immediate early genes (IEG), transcription factors, cytokines, and membrane receptors were significantly up-regulated by LTB(4). LTB(4) and LTD(4) had similar effects on signaling, gene expression, and chemotaxis indicating redundant cell activation pathways but costimulation with both lipid mediators was additive for many monocyte functions. CONCLUSION:Leukotriene B(4) and LTD(4) display both redundant and cooperative effects on intracellular signaling, gene expression, and chemotaxis in human monocytes. These findings suggest that therapies targeting either leukotriene alone may be less effective than approaches directed at both.

| S-EPMC3170431 | biostudies-literature

Leukotriene D4 induces early genes in Hodgkin lymphoma cells through Cysteinyl leukotriene type I receptor

Project description:Leukotriene (LT) D4 contributes to aberrant cytokine networks of classical Hodgkin lymphonoma, This study has investigated LTD4 induced gene expression and pathway in Hodgkin lymphonma cell line L1236.

2015-01-01 | GSE43855 | GEO

Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes

Project description:LTB4, 50 nmol/L for 30 minutes, induced expression of 27 genes in cultured human elutriated monocytes comparred to vehicle (ethanol) treated control cells.

2011-09-21 | GSE24869 | GEO

GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4.

Project description:The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT(1)R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D(4) with stable transfectants. However, in stable cotransfections of 6xHis-tagged GPR17 with Myc-tagged CysLT(1)R, the robust CysLT(1)R-mediated calcium response to LTD(4) was abolished. The membrane expression of the CysLT(1)R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD(4)-elicited ERK phosphorylation and the specific binding of [(3)H]LTD(4) to microsomal membranes were fully inhibited. CysLT(1)R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT(1)R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMPhis) increased both the membrane expression of CysLT(1)R protein by FACS analysis and the LTD(4)-elicited calcium flux in a dose-dependent manner as compared with control BMMPhis, indicating a negative regulatory function of GPR17 for CysLT(1)R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT(1)R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT(1)R that suppresses CysLT(1)R-mediated function at the cell membrane.

| S-EPMC2710631 | biostudies-literature

Leukotriene D4 induces gene expression in human monocytes through cysteinyl leukotriene type I receptor.

Project description:Background: Cysteinyl leukotrienes (cysLTs) are important mediators of innate immune responsiveness and chronic inflammatory diseases. CysLTs acting through cysteinyl leukotriene receptors may influence the migration and activity of cells such as eosinophils, monocytes and dendritic cells. Objective: To determine the gene expression signature of human monocytes in response to cysLTs and to elucidate the signaling pathways involved in monocyte activation. Methods: Gene expression was analyzed using oligonucleotide microarrays. Responsiveness to cysLTs was assessed by real-time PCR, calcium flux, kinase activation and chemotaxis assays. Results: Cysteinyl leukotriene type I receptor (CysLTR1) transcript 1 is predominantly expressed in human monocytes and cysLTs signal through CysLTR1 in these cells. Several immediate-early genes, including early growth response (Egr) -2, 3, FosB, activating transcription factor 3 and nuclear receptor subfamily 4 were significantly induced by LTD4. This effect was mediated by CysLTR1 coupled to Gαi/o, activation of phospholipase C, and inositol-1,4,5-triphosphate (IP3) and store operated calcium channels. LTD4 induced p38 MAP kinase phosphorylation, a pathway also involved in the regulation of immediate-early genes expression in monocytes. LTD4 stimulated monocyte chemotactic activity that was fully blocked by a selective CysLTR1 inhibitor MK571 and pertussis toxin, suggesting that CysLTR1 coupled to Gαi/o is a dominant functional pathway in human monocytes. Conclusion: Our data show that cysLTs acting through CysLTR1 can significantly influence the activation and migration of human monocytes and that these effects can be fully inhibited by CysLTR1 antagonists. Clinical implications: Antileukotriene therapies are likely to significantly block the proinflammatory functions of human monocytes. Keywords: monocytes stimulated with LTD4

2007-08-30 | GSE7807 | GEO

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