Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells.

Nuclear factor of activated T cells 5 (NFAT5) and cyclooxygenase 2 (COX2; PTGS2) both participate in diverse pathologies including cancer progression. However, the biological role of the NFAT5-COX2 signaling pathway in human endometrial cancer has remained elusive. The present study explored whether NFAT5 is expressed in endometrial tumors and if NFAT5 participates in cancer progression. To gain insights into the underlying mechanisms, NFAT5 protein abundance in endometrial cancer tissue was visualized by immunohistochemistry and endometrial cancer cells (Ishikawa and HEC1a) were transfected with NFAT5 or with an empty plasmid. As a result, NFAT5 expression is more abundant in high-grade than in low-grade endometrial cancer tissue. RNA sequencing analysis of NFAT5 overexpression in Ishikawa cells upregulated 37 genes and downregulated 20 genes. Genes affected included cyclooxygenase 2 and hypoxia inducible factor 1α (HIF1A). NFAT5 transfection and/or treatment with HIF-1α stabilizer exerted a strong stimulating effect on HIF-1α promoter activity as well as COX2 expression level and prostaglandin E2 receptor (PGE2) levels. Our findings suggest that activation of NFAT5-HIF-1α-COX2 axis could promote endometrial cancer progression.

Glucose transport in lymphocytes.

Glucose uptake into lymphocytes is accomplished by non-concentrative glucose carriers of the GLUT family (GLUT1, GLUT3, GLUT4, GLUT6) and/or by the Na+-coupled glucose carrier SGLT1. The latter accumulates glucose against glucose gradients and is still effective at very low extracellular glucose concentrations. Signaling involved in SGLT1 expression and activity includes protein kinase A (PKA), protein kinase C (PKC), serum- and glucocorticoid-inducible kinase (SGK1), AMP-activated kinase (AMPK), and Janus kinases (JAK2 and JAK3). Glucose taken up is partially stored as glycogen. In hypoxic environments, such as in tumors as well as infected and inflamed tissues, lymphocytes depend on energy production from glycogen-dependent glycolysis. The lack of SGLT1 may compromise glycogen storage and thus lymphocyte survival and function in hypoxic tissues. Accordingly, in mice, genetic knockout of sglt1 compromised bacterial clearance following Listeria monocytogenes infection leading to an invariably lethal course of the disease. Whether the effect was due to the lack of sglt1 in lymphocytes or in other cell types still remains to be determined. Clearly, additional experimental effort is required to define the role of glucose transport by GLUTs and particularly by SGLT1 for lymphocyte survival and function, as well as orchestration of the host defense against tumors and bacterial infections.

The Putative Role of 1,25(OH)2D3 in the Association of Milk Consumption and Parkinson's Disease.

The consumption of dairy products, particularly of low fat milk, has been shown to be associated with the occurrence of Parkinson's disease. This association does not necessarily reflect a pathophysiological role of milk intake in the development of Parkinson's disease. Nevertheless, the present review discusses a potential mechanism possibly mediating an effect of milk consumption on Parkinson's disease. The case is made that milk is tailored in part to support bone mineralization of the suckling offspring and is thus rich in calcium and phosphate. Milk intake is thus expected to enhance intestinal calcium phosphate uptake. As binding to fatty acids impedes Ca2+ absorption, low fat milk is particularly effective. Calcium and phosphate uptake inhibit the formation of 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), the active form of vitamin D. Calcium inhibits 1,25(OH)2D3 production in part by suppressing the release of parathyroid hormone, a powerful stimulator of 1,25(OH)2D3 formation. Phosphate excess stimulates the release of fibroblast growth factor FGF23, which suppresses 1,25(OH)2D3 formation, an effect requiring Klotho. 1,25(OH)2D3 is a main regulator of mineral metabolism, but has powerful effects apparently unrelated to mineral metabolism, including suppression of inflammation and influence of multiple brain functions. In mice, lack of 1,25(OH)2D3 and excessive 1,25(OH)2D3 formation have profound effects on several types of behavior, such as explorative behavior, anxiety, grooming and social behavior. 1,25(OH)2D3 is produced in human brain and influences the function of various structures including substantia nigra. In neurons 1,25(OH)2D3 suppresses oxidative stress, inhibits inflammation and stimulates neurotrophin formation thus providing neuroprotection. As a result, 1,25(OH)2D3 is considered to favorably influence the clinical course of Parkinson's disease. In conclusion, consumption of milk could in theory accelerate the downhill course of neuronal function in Parkinson's disease. However, substantial additional experimentation is required to define the putative causal role of 1,25(OH)2D3 in the pathophysiology of Parkinson's disease and its sensitivity to milk consumption.

The Putative Role of 1,25(OH)2D3 in the Association of Milk Consumption and Parkinson's Disease.

The consumption of dairy products, particularly of low fat milk, has been shown to be associated with the occurrence of Parkinson's disease. This association does not necessarily reflect a pathophysiological role of milk intake in the development of Parkinson's disease. Nevertheless, the present review discusses a potential mechanism possibly mediating an effect of milk consumption on Parkinson's disease. The case is made that milk is tailored in part to support bone mineralization of the suckling offspring and is thus rich in calcium and phosphate. Milk intake is thus expected to enhance intestinal calcium phosphate uptake. As binding to fatty acids impedes Ca2+ absorption, low fat milk is particularly effective. Calcium and phosphate uptake inhibit the formation of 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), the active form of vitamin D. Calcium inhibits 1,25(OH)2D3 production in part by suppressing the release of parathyroid hormone, a powerful stimulator of 1,25(OH)2D3 formation. Phosphate excess stimulates the release of fibroblast growth factor FGF23, which suppresses 1,25(OH)2D3 formation, an effect requiring Klotho. 1,25(OH)2D3 is a main regulator of mineral metabolism, but has powerful effects apparently unrelated to mineral metabolism, including suppression of inflammation and influence of multiple brain functions. In mice, lack of 1,25(OH)2D3 and excessive 1,25(OH)2D3 formation have profound effects on several types of behavior, such as explorative behavior, anxiety, grooming and social behavior. 1,25(OH)2D3 is produced in human brain and influences the function of various structures including substantia nigra. In neurons 1,25(OH)2D3 suppresses oxidative stress, inhibits inflammation and stimulates neurotrophin formation thus providing neuroprotection. As a result, 1,25(OH)2D3 is considered to favorably influence the clinical course of Parkinson's disease. In conclusion, consumption of milk could in theory accelerate the downhill course of neuronal function in Parkinson's disease. However, substantial additional experimentation is required to define the putative causal role of 1,25(OH)2D3 in the pathophysiology of Parkinson's disease and its sensitivity to milk consumption.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Alkaline Cytosolic pH and High Sodium Hydrogen Exchanger 1 (NHE1) Activity in Th9 Cells.

CD4+ T helper 9 (Th9) cells are a newly discovered Th cell subset that produce the pleiotropic cytokine IL-9. Th9 cells can protect against tumors and provide resistance against helminth infections. Given their pivotal role in the adaptive immune system, understanding Th9 cell development and the regulation of IL-9 production could open novel immunotherapeutic opportunities. The Na+/H+ exchanger 1 (NHE1; gene name Slc9α1)) is critically important for regulating intracellular pH (pHi), cell volume, migration, and cell survival. The pHi influences cytokine secretion, activities of membrane-associated enzymes, ion transport, and other effector signaling molecules such as ATP and Ca2+ levels. However, whether NHE1 regulates Th9 cell development or IL-9 secretion has not yet been defined. The present study explored the role of NHE1 in Th9 cell development and function. Th cell subsets were characterized by flow cytometry and pHi was measured using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) dye. NHE1 functional activity was estimated from the rate of realkalinization following an ammonium pulse. Surprisingly, in Th9 cells pHi and NHE1 activity were significantly higher than in all other Th cell subsets (Th1/Th2/Th17 and induced regulatory T cells (iTregs)). NHE1 transcript levels and protein abundance were significantly higher in Th9 cells than in other Th cell subsets. Inhibition of NHE1 by siRNA-NHE1 or with cariporide in Th9 cells down-regulated IL-9 and ATP production. NHE1 activity, Th9 cell development, and IL-9 production were further blunted by pharmacological inhibition of protein kinase Akt1/Akt2. Our findings reveal that Akt1/Akt2 control of NHE1 could be an important physiological regulator of Th9 cell differentiation, IL-9 secretion, and ATP production.

DJ-1/Park7 Sensitive Na+ /H+ Exchanger 1 (NHE1) in CD4+ T Cells.

DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+ /H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pHi ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ-1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4+ T cells, and blunted the difference between DJ-1-/- and DJ-1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1-/- CD4+ T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc.

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells.

BACKGROUND/AIMS: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. METHODS: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. RESULTS: A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. CONCLUSION: Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth inhibition of tumor cells.

1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells.

BACKGROUND/AIMS: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. METHODS: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. RESULTS: A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. CONCLUSIONS: In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)2D3 stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells.

BACKGROUND: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. METHODS: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. RESULTS: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. CONCLUSIONS: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

SGLT1 Deficiency Turns Listeria Infection into a Lethal Disease in Mice.

BACKGROUND: Cellular glucose uptake may involve either non-concentrative glucose carriers of the GLUT family or Na+-coupled glucose-carrier SGLT1, which accumulates glucose against glucose gradients and may thus accomplish cellular glucose uptake even at dramatically decreased extracellular glucose concentrations. SGLT1 is not only expressed in epithelia but as well in tumour cells and immune cells. Immune cell functions strongly depend on their metabolism, therefore we hypothesized that deficiency of SGLT1 modulates the defence against bacterial infection. To test this hypothesis, we infected wild type mice and gene targeted mice lacking functional SGLT1 with Listeria monocytogenes. METHODS: SGLT1 deficient mice and wild type littermates were infected with 1x104 CFU Listeria monocytogenes intravenously. Bacterial titers were determined by colony forming assay, SGLT1, TNF-α, IL-6 and IL-12a transcript levels were determined by qRT-PCR, as well as SGLT1 protein abundance and localization by immunohistochemistry. RESULTS: Genetic knockout of SGLT1 (Slc5a1-/- mice) significantly compromised bacterial clearance following Listeria monocytogenes infection with significantly enhanced bacterial load in liver, spleen, kidney and lung, and significantly augmented hepatic expression of TNF-α and IL-12a. While all wild type mice survived, all SGLT1 deficient mice died from the infection. CONCLUSIONS: SGLT1 is required for bacterial clearance and host survival following murine Listeria infection.

Decidualization is Impaired in Endometrial Stromal Cells from Uterine Rudiments in Mayer-Rokitansky-Küster-Hauser Syndrome.

BACKGROUND/AIMS: Uterine rudiments from patients with Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) contain all tissues typically found in the uterus. Endometrium from the rudiments predominantly exhibits basalis-like features, and endometrial proliferative capacity in patients' epithelium and stroma is significantly lower. METHODS: This single-center, prospective study conducted at a major German university hospital compared in-vitro decidualization in cultured ESCs from MRKHS patients and hysterectomy controls. Primary ESC cultures were established from both sources. Hormone-induced prolactin and IGFBP-1 secretion served as a measure of their ability to undergo decidualization in response to hormonal stimulation. Expression levels of 8 key marker genes of decidualization were also determined. RESULTS: At day 9, mean secretion of prolactin and IGFBP-1 was significantly reduced by 89.0% and 99.5%, respectively, in MRKHS ESCs vs. hysterectomy controls, both indicating impaired decidualization of MRKHS ESCs. Key decidual markers confirmed impaired decidualization in MRKHS patients. CONCLUSION: Our results indicate that the ESCs from MRKHS patients lack hormone responsiveness as a potential sign of dysfunctional hormone receptor function, which may also play a role in the onset of MRKHS. Further studies are needed to corroborate our findings, directly address receptor function, and elucidate the role of other potential determinants of uterine development and adult function.

ß-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2.

BACKGROUND/AIMS: ß-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a ß-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of ß-Klotho protein on the peptide transporters PEPT1 and PEPT2. METHODS: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of ß-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. RESULTS: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble ß-Klotho protein. As shown for PEPT1, ß-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of ß-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. CONCLUSION: ß-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a.

BACKGROUND: LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. AIMS: The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. METHODS: FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. RESULTS: LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. CONCLUSION: In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration.

1α,25(OH)2D3 Induces Actin Depolymerization in Endometrial Carcinoma Cells by Targeting RAC1 and PAK1.

BACKGROUND: Cell proliferation and motility require actin reorganization, which is under control of various signalling pathways including ras-related C3 botulinum toxin substrate 1 (RAC1), p21 protein-activated kinase 1 (PAK1) and actin related protein 2 (ARP2). Tumour cell proliferation is modified by 1α,25-Dihydroxy-Vitamin D3 (1α,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. The present study explored whether 1α,25(OH)2D3 modifies actin cytoskeleton in Ishikawa cells, a well differentiated endometrial carcinoma cell line. METHODS: To this end, actin cytoskeleton was visualized by confocal microscopy. Globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry, transcript levels by qRT-PCR and protein abundance by immunoblotting. RESULTS: A 24 hour treatment with 1α,25(OH)2D3 (100 nM) significantly decreased RAC1 and PAK1 transcript levels and activity, decreased ARP2 protein levels and depolymerized actin. The effect of 1α,25(OH)2D3 on actin polymerization was mimicked by pharmacological inhibition of RAC1 and PAK1. CONCLUSIONS: 1α,25(OH)2D3 leads to disruption of RAC1 and PAK1 activity with subsequent actin depolymerization of endometrial carcinoma cells.

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

BACKGROUND/AIMS: Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. METHODS: Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. RESULTS: ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-ß and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1ß, IL-6 and IL-17 but not IFN-γ mRNA levels. CONCLUSION: ASM is a negative regulator of natural and iTreg.

Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation.

BACKGROUND: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation. METHODS/RESULTS: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites. CONCLUSION: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.

LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1.

BACKGROUND: Serum & glucocorticoid inducible kinase (SGK1) regulates several ion channels, including amiloride sensitive epithelial Na+ channel (ENaC). SGK1 and ENaC in the luminal endometrium epithelium, are critically involved in embryo implantation, although little is known about their regulation. The present study explored whether SGK1 and ENaC are modulated by LEFTYA, a negative regulator of uterine receptivity. METHODS: Expression levels were determined by qRT-PCR and Western blotting, ENaC channel activity by whole cell patch clamp and transepithelial current by Ussing chamber experiments. RESULTS: Treatment of Ishikawa cells, an endometrial adenocarcinoma model cell line of endometrial epithelial cells, with LEFTYA rapidly up-regulated SGK1 and ENaC transcript and protein levels. Induction of ENaC in response to LEFTYA was blunted upon co-treatment with the SGK1 inhibitor EMD638683. ENaC levels also significantly upregulated upon expression of a constitutively active, but not a kinase dead, SGK1 mutant in Ishikawa cells. LEFTYA increased amiloride sensitive Na+-currents in Ishikawa cells and amiloride sensitive transepithelial current across the murine endometrium. Furthermore, LEFTYA induced the expression of ENaC in the endometrium of wild-type but not of Sgk1-deficient mice. CONCLUSIONS: LEFTYA regulates the expression and activity of ENaC in endometrial epithelial cells via SGK1. Aberrant regulation of SGK1 and ENaC by LEFTYA could contribute to the pathogenesis of unexplained infertility.

Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells.

BACKGROUND/AIMS: Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions. METHODS: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4. RESULTS: We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice. CONCLUSION: Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions.

Up-regulation of FGF23 release by aldosterone.

The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry.