scholarly journals Activation of Type 1 CRH Receptor Isoforms Induces Serotonin Release from Human Carcinoid BON-1N Cells: An Enterochromaffin Cell Model

Endocrinology ◽  
2011 ◽  
Vol 152 (1) ◽  
pp. 126-137 ◽  
Author(s):  
S. Vincent Wu ◽  
Pu-Qing Yuan ◽  
Jim Lai ◽  
Kelvin Wong ◽  
Monica C. Chen ◽  
...  
Keyword(s):  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Splice Variants ◽  
Cell Model ◽  
Serotonin Release ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Maximal Increase ◽  
Ec Cells

Abstract CRH and 5-hydroxytryptamine (5-HT) are expressed in human colonic enterochromaffin (EC) cells, but their interactions at the cellular level remain largely unknown. The mechanistic and functional relationship between CRH and 5-HT systems in EC cells was investigated in a human carcinoid cloned BON cell line (BON-1N), widely used as an in vitro model of EC cell function. First, we identified multiple CRH1 splice variants, including CRH1a, CRH1c, CRH1f, and a novel form lacking exon 4, designated here as CRH1i, in the BON-1N cells. The expression of CRH1i was also confirmed in human brain cortex, pituitary gland, and ileum. Immunocytochemistry and immunoblot analysis confirmed that BON-1N cells were CRH1 and 5-HT positive. CRH, urocortin (Ucn)-1, and cortagine, a selective CRH1 agonist, all increased intracellular cAMP, and this concentration-dependent response was inhibited by CRH1-selective antagonist NBI-35965. CRH and Ucn-1, but not Ucn-2, stimulated significant ERK1/2 phosphorylation. In transfected human embryonic kidney-293 cells, CRH1i isoforms produced a significant increase in pERK1/2 in response to CRH1 agonists that was sensitive to NBI-35965. CRH and Ucn-1 stimulated 5-HT release that reached a maximal increase of 3.3- and 4-fold at 10−8m over the basal level, respectively. In addition, exposure to CRH for 24-h up-regulated tryptophan hydroxylase-1 mRNA levels in the BON-1N cells. These findings define the expression of EC cell-specific CRH1 isoforms and activation of CRH1-dependent pathways leading to 5-HT release and synthesis; thus, providing functional evidence of a link exists between CRH and 5-HT systems, which have implications in stress-induced CRH1 and 5-HT-mediated stimulation of lower intestinal function.

scholarly journals The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

2012 ◽  
Vol 302 (3) ◽  
pp. G397-G405 ◽  
Author(s):  
A. Chin ◽  
B. Svejda ◽  
B. I. Gustafsson ◽  
A. B. Granlund ◽  
A. K. Sandvik ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Cell System ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Serotonin Secretion ◽  
A2b Receptor ◽  
Ec Cells ◽  
Inflammatory Bowel

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98–99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10−6 M; IC50 = 3.7 × 10−8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

scholarly journals Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

2007 ◽  
Vol 38 (1) ◽  
pp. 181-192 ◽  
Author(s):  
Mark Kidd ◽  
Geeta N Eick ◽  
Irvin M Modlin ◽  
Roswitha Pfragner ◽  
Manish C Champaneria ◽  
...  
Keyword(s):  
Substance P ◽  
Cell Line ◽  
Tryptophan Hydroxylase ◽  
Intracellular Camp ◽  
Cell Of Origin ◽  
Rt Pcr ◽  
Cytoplasmic Vesicles ◽  
Ec Cells ◽  
Gastrointestinal Carcinoid

Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic α1C and β1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC50=340 nM) and 5-HT (EC50=81 nM) which was completely inhibited by the cAMP antagonist 2′,5′-dideoxyadenosine (10 μM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC50=420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20±12 and 35±5% respectively) in serum-free medium whereas gefitinib (1 nM–10 μM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

scholarly journals YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

2021 ◽  
Vol 22 (13) ◽  
pp. 7226
Author(s):  
Violeta Stojanovska ◽  
Aneri Shah ◽  
Katja Woidacki ◽  
Florence Fischer ◽  
Mario Bauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Trophoblast Cells ◽  
Invasion And Migration ◽  
And Migration ◽  
Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

scholarly journals Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro

2021 ◽  
Author(s):  
Jens Weusmann ◽  
James Deschner ◽  
Jean-Claude Imber ◽  
Anna Damanaki ◽  
Natalia D. P. Leguizamón ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Function ◽  
Nuclear Translocation ◽  
In Vitro Study ◽  
Human Gingival Fibroblasts ◽  
Mrna Levels ◽  
Gingival Fibroblasts ◽  
Air Polishing ◽  
Wide Range

Abstract Objectives Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. Methods HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett’s and Tukey’s tests (p < 0.05). Results Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. Conclusion Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder.

Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer

2020 ◽  
Author(s):  
Ben Yang ◽  
Wang Ke ◽  
Yingchun Wan ◽  
Tao Li
Keyword(s):  
Endometrial Cancer ◽  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Mice Model ◽  
Expression Levels ◽  
Gynecological Malignancy ◽  
Ec Cells

Abstract Background Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major difficulties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. Methods Cisplatin and doxorubicin resistant cell lines were acquired by continuous exposing parental EC cells to cisplatin or doxorubicin for 3 months. Cell viability was determined by using MTT assay. Protein Expression levels of protein were examined by western blotting assay. mRNA levels were measured by quantitative polymerase chain reaction (qPCR) assay. Ring finger protein 8 (RNF8) knockout cell lines were generated by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay. Nonhomologous end joining (NHEJ) efficiency were quantified by plasmid based NHEJ assay. DNA double strand breaks (DSB) were generated using laser micro-irradiation. Protein recruitment to DSB was analyzed by immunofluorescent assay. Tumor growth was examined by AN3CA xenograft mice model. Results We found that protein and mRNA expression levels of RNF8 were significantly increased in both cisplatin and doxorubicin resistant EC cells. Cell survival assay showed that RNF deficiency significantly enhanced the sensitivity of resistant EC cells to cisplatin and doxorubicin (P < 0.01). In addition, chemoresistant EC cells exhibited increased NHEJ efficiency. Knockout of RNF8 in chemoresistant EC cells significantly reduced NHEJ efficiency and prolonged Ku80 retention on DSB. Moreover, cisplatin resistant AN3CA xenograft showed that RNF8 deficiency overcame cisplatin resistance. Conclusions Our in vitro and in vivo assays provide evidence for RNF8, which is a NHEJ factor, serving as a promising, novel target in EC chemotherapy.

scholarly journals Starch Digestion Enhances Bioaccessibility of Anti-Inflammatory Polyphenols from Borlotti Beans (Phaseolus vulgaris)

Nutrients ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 295
Author(s):  
Perez-Hernandez ◽  
Nugraheni ◽  
Benohoud ◽  
Sun ◽  
Hernández-Álvarez ◽  
...  
Keyword(s):  
Phaseolus Vulgaris ◽  
Cell Model ◽  
In Vitro Digestion ◽  
Enzymatic Digestion ◽  
Mrna Levels ◽  
Starch Digestion ◽  
Granule Structure ◽  
Domestic Processing ◽  
Anti Inflammatory

The consumption of beans has been associated with chronic disease prevention which may be attributed to the polyphenols present in the seed coat and endosperm. However, their bioaccessibility is likely to be limited by interactions with bean matrix components, including starch, protein and fibre. The aim of this project was to evaluate the effect of domestic processing and enzymatic digestion on the bioaccessibility of polyphenols from Borlotti beans (Phaseolus vulgaris) and to test their anti-inflammatory properties in a macrophage cell model. In vitro digestion of cooked beans released twenty times more polyphenols (40.4 ± 2.5 mg gallic acid equivalents (GAE)/g) than domestic processing (2.22 ± 0.1 mg GAE/g), with starch digestion contributing to the highest release (30.9 ± 0.75 mg GAE/g). Fluorescence microscopy visualization of isolated bean starch suggests that polyphenols are embedded within the granule structure. LC-MS analysis showed that cooked Borlotti bean contain flavonoids, flavones and hydroxycinnamic acids, and cooked bean extracts exerted moderate anti-inflammatory effects by decreasing mRNA levels of IL1β and iNOS by 25% and 40%, respectively. In conclusion, the bioaccessibility of bean polyphenols is strongly enhanced by starch digestion. These polyphenols may contribute to the health benefits associated with bean consumption.

Luminal regulation of normal and neoplastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants

2008 ◽  
Vol 295 (2) ◽  
pp. G260-G272 ◽  
Author(s):  
Mark Kidd ◽  
Irvin M. Modlin ◽  
Bjorn I. Gustafsson ◽  
Ignat Drozdov ◽  
Oyvind Hauso ◽  
...  
Keyword(s):  
Bile Salts ◽  
Cell Function ◽  
Sodium Deoxycholate ◽  
Serotonin Release ◽  
Artificial Sweetener ◽  
Cell Regulation ◽  
Analog Ic ◽  
Erk Phosphorylation ◽  
Antisense Knockdown ◽  
Ec Cells

Mechanisms by which gut luminal content regulates secretion and motility are ill understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Pure (98–99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) G protein-coupled receptors (GPCRs) and transporters for glutamine (SNAT1/2), glucose (GLUT1/3/SGLT1), and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5-HT release (EC50 = 0.002–0.2 μM; 2-fold release) but were 10–100 times more potent in neoplastic EC cells, which also secreted 6–13 times more 5-HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol and eugenol) also stimulated normal and neoplastic EC cell 5-HT secretion (EC50 = 1.2 nM to 2.1 μM and 0.05 nM to 0.1 μM release, respectively); 2-deoxyglucose and the artificial sweetener sucralose also stimulated (EC50 = 9.2 and 0.38 nM). 5-HT release was associated with ERK phosphorylation (1.5-fold, P < 0.02) and could be inhibited by a somatostatin analog (IC50 = 1 pM). Eleven secretory associated genes including the vesicle docking inhibitor STXBP3 were upregulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression by use of antisense knockdown significantly ( P < 0.05) reduced 5-HT secretion. In conclusion, EC cells express GPCRs and transporters for luminal tastants, olfactants, glutamine, glucose, and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation, and 5-HT secretion. Luminal EC cell regulation is likely to be as important as G cell regulation in gastric acid secretion; development of agents to target EC cell function is therefore a critical therapeutic goal.

scholarly journals Liver X receptor agonist TO-901317 upregulates SCD1 expression in renal proximal straight tubule

2006 ◽  
Vol 290 (5) ◽  
pp. F1065-F1073 ◽  
Author(s):  
Yahua Zhang ◽  
Xiaoyan Zhang ◽  
Lihong Chen ◽  
Jing Wu ◽  
Dongming Su ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Cell Function ◽  
Regulatory Element ◽  
Cholesterol Absorption ◽  
Mrna Levels ◽  
Double Labeling ◽  
Straight Tubules ◽  
Scd1 Expression

Liver X receptors (LXRs), including LXRα and LXRβ, are intracellular sterol sensors that regulate expression of genes controlling fatty acid and cholesterol absorption, excretion, catabolism, and cellular efflux. Because the kidney plays an important role in lipid metabolism and dyslipidemia accelerates renal damage, we investigated the effect of TO-901317, an LXR agonist, on the gene expression profile in mouse kidney. Treatment of C57 Bl/6 mice with TO-901317 (3 mg·kg−1·day−1) for 3 days resulted in 51 transcripts that were significantly regulated in the kidney. Among them, the stearoyl-CoA desaturase-1 (SCD1) was upregulated most dramatically. Northern blot analysis revealed that SCD1 mRNA levels were markedly higher than that in control kidneys. Enhanced SCD1 expression by TO-901317 also resulted in increased fatty acid desaturation in the kidney. In control mice, constitutive renal SCD1 expression was low; however, TO-901317 treatment markedly increased SCD1 expression in the outer stripe of the outer medulla as assessed by both in situ hybridization and immunostain. Double-labeling studies further indicated that SCD1 mRNA was selectively expressed in proximal straight tubules negative for aquaporin-2 and Tamm-Horsfall protein. In vitro studies in cultured murine proximal tubule cells further demonstrated that LXR activation enhanced SCD1 transcription via increased sterol regulatory element binding protein-1. Taken together, these data suggest LXR activation of SCD1 expression may play an important role in regulating lipid metabolism and cell function in renal proximal straight tubules.

scholarly journals Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms.

1992 ◽  
Vol 12 (6) ◽  
pp. 2681-2689 ◽  
Author(s):  
E M Douville ◽  
D E Afar ◽  
B W Howell ◽  
K Letwin ◽  
L Tannock ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Expression Cloning ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Intracellular Enzyme ◽  
Exogenous Substrate ◽  
Ec Cells ◽  
Membrane Spanning

A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.

scholarly journals IL-10 is up-regulated in multiple cell types during viremic HIV infection and reversibly inhibits virus-specific T cells

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 346-356 ◽  
Author(s):  
Mark A. Brockman ◽  
Douglas S. Kwon ◽  
Daniel P. Tighe ◽  
David F. Pavlik ◽  
Pamela C. Rosato ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Cell Types ◽  
Interleukin 10 ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Multiple Cell

AbstractMurine models indicate that interleukin-10 (IL-10) can suppress viral clearance, and interventional blockade of IL-10 activity has been proposed to enhance immunity in chronic viral infections. Increased IL-10 levels have been observed during HIV infection and IL-10 blockade has been shown to enhance T-cell function in some HIV-infected subjects. However, the categories of individuals in whom the IL-10 pathway is up-regulated are poorly defined, and the cellular sources of IL-10 in these subjects remain to be determined. Here we report that blockade of the IL-10 pathway augmented in vitro proliferative capacity of HIV-specific CD4 and CD8 T cells in individuals with ongoing viral replication. IL-10 blockade also increased cytokine secretion by HIV-specific CD4 T cells. Spontaneous IL-10 expression, measured as either plasma IL-10 protein or IL-10 mRNA in peripheral blood mononuclear cells (PBMCs), correlated positively with viral load and diminished after successful antiretroviral therapy. IL-10 mRNA levels were up-regulated in multiple PBMC subsets in HIV-infected subjects compared with HIV-negative controls, particularly in T, B, and natural killer (NK) cells, whereas monocytes were a major source of IL-10 mRNA in HIV-infected and -uninfected individuals. These data indicate that multiple cell types contribute to IL-10–mediated immune suppression in the presence of uncontrolled HIV viremia.

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