scholarly journals Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A549-A549
Author(s):  
Laura C Hernández-Ramírez ◽  
Nathan Pankratz ◽  
John Lane ◽  
Mingming Hu ◽  
Fabio R Faucz ◽  
...  
Keyword(s):  
Cell Line ◽  
Intracellular Signaling ◽  
Mrna Level ◽  
Disease Onset ◽  
Digital Pcr ◽  
Hek293 Cells ◽  
Parental Cell Line ◽  
Nucleotide Exchange ◽  
Wild Type

Abstract Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.

scholarly journals Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC-resistant rat leukemic cell line of clonal origin fully restores drug sensitivity

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1188-1194 ◽  
Author(s):  
AP Stegmann ◽  
WH Honders ◽  
R Willemze ◽  
VW Ruiz van Haperen ◽  
JE Landegent
Keyword(s):  
Cell Line ◽  
Leukemic Cell ◽  
Polymerase Chain Reaction Analysis ◽  
Deoxycytidine Kinase ◽  
Target Cells ◽  
Parental Cell Line ◽  
Wild Type ◽  
Coding Region ◽  
Leukemic Cell Line

The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro-induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1-ADCK) that were stably transfected and monitored over a period of 14 weeks. Northern and Western blot analysis showed restoration of dck mRNA and protein expression. Initial rate measurements of DCK activity showed that Km values for dck were only slightly altered as a result of transfection, whereas strongly increased Vmax values were observed, resulting in a 12-fold increased phosphorylation efficiency for both dC and AraC, compared with the AraC-sensitive parental cell line RO/1 from which the RO/1-A was originally derived. In vitro sensitivity to AraC- and DAC-mediated cytotoxicity was fully restored in RO/1-ADCK. The data pinpoint acquired DCK deficiency caused by mutations of the dck gene as the major cause of AraC resistance in this model.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Abstract 14035: Renal Tubular Secretion and Cardiac Distribution of Dofetilide is Dependent on MATE1 Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Muhammad Erfan Uddin ◽  
Yan Jin ◽  
Alice A Gibson ◽  
Ingrid M Bonilla ◽  
Cynthia A Carnes ◽  
...  
Keyword(s):  
Organic Cation ◽  
Tubular Secretion ◽  
Hek293 Cells ◽  
Delayed Rectifier ◽  
Organic Cation Transporters ◽  
Wild Type ◽  
Renal Tubular Secretion ◽  
Cation Transporters ◽  
Renal Tubular

Introduction: Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients with atrial fibrillation and flutter, and its use is associated with a risk of QT prolongation and Torsades de Pointes . The mechanisms involved in dofetilide’s renal tubular secretion and its uptake into cardiomyocytes remain unknown. Previously reported drug-drug interaction (DDI) studies suggest the involvement of organic cation transporters. Here, we investigated the contribution of organic cation transporters (OCT2 and MATE1) to the pharmacokinetics of dofetilide to gain insight into its DDI potential. Hypothesis: Based on known DDIs with dofetilide, we hypothesize that OCT2 and/or MATE1 play a key role in the inter-individual variability in pharmacokinetics and pharmacodynamics of dofetilide. Methods: In vitro and ex vivo transport kinetics of dofetilide were determined in HEK293 cells stably transfected with OCT2 or MATE1, and in isolated cardiomyocytes, respectively. In vivo studies were performed in wild-type, OCT2-, and MATE1-deficient mice (n=5) receiving dofetilide (5 mg/kg, p.o., 2.5 mg/kg, i.v.), with or without several contraindicated drugs. Dofetilide concentrations in plasma and urine were determined by UPLC-MS/MS. Results: In vitro studies demonstrated that dofetilide is a good substrate of MATE1 but not OCT2. Deficiency of MATE1 was associated with increased plasma concentrations of dofetilide and with a significantly reduced urinary excretion (3-fold in females and 5-fold in males, respectively). Dofetilide accumulation in cardiomyocytes was increased by 2-fold in MATE1-deficient females, and pre-incubation with the MATE1 inhibitor cimetidine significantly reduced dofetilide uptake in wild-type cardiomyocytes. Several contraindicated drugs listed in the dofetilide prescribing information, including cimetidine, ketoconazole, increased dofetilide plasma exposure in wild-type mice by >2.8-fold. Conclusion: Renal secretion of dofetilide is mediated by MATE1 and is highly sensitive to inhibition by many widely used prescription drugs that can cause clinically relevant DDIs. Deficiency of MATE1 also increases accumulation in the heart which may contribute to individual variation in response to dofetilide.

scholarly journals Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts.

1996 ◽  
Vol 132 (1) ◽  
pp. 181-193 ◽  
Author(s):  
S Yoshida ◽  
A Fujisawa-Sehara ◽  
T Taki ◽  
K Arai ◽  
Y Nabeshima
Keyword(s):  
Lysophosphatidic Acid ◽  
Intracellular Signaling ◽  
Myogenic Differentiation ◽  
Mrna Level ◽  
Biological Significance ◽  
Cell Contact ◽  
C2c12 Myoblast ◽  
Bhlh Proteins ◽  
Cell Cell

Myogenic cells provide excellent in vitro models for studying the cell growth and differentiation. In this study we report that lysophosphatidic acid (LPA), a bioactive phospholipid contained in serum, stimulates the growth and inhibits the differentiation of mouse C2C12 myoblast cells, in a distinct manner from basic fibroblast growth factor (bFGF) whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were both blocked by pertussis toxin, suggesting the involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases. Detailed analysis revealed that LPA and bFGF act differently in regulating the myogenic basic helix-loop-helix (bHLH) proteins, the key players in myogenic differentiation process. LPA stimulates the proliferation of undifferentiated myoblasts allowing the continued expression of MyoD, but in contrast, bFGF does so with the MyoD expression suppressed at the mRNA level. Both compounds maintain the myf-5 expression, and suppress the myogenin expression. In addition, while LPA did not inhibit cell-cell contact-induced differentiation, bFGF strongly inhibited this process. Furthermore, LPA and bFGF act cooperatively in their mitogenic and anti-differentiation abilities. These findings indicate that LPA and bFGF differently stimulate intracellular signaling pathways, resulting in proliferating myoblasts each bearing a distinct expression pattern of myogenic bHLH proteins and distinct differentiation potentials in response to cell-cell contact, and illustrate the biological significance of Gi-mediated and tyrosine kinase-mediated signals.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

2009 ◽  
Vol 83 (18) ◽  
pp. 9079-9093 ◽  
Author(s):  
Laura Pacini ◽  
Rita Graziani ◽  
Linda Bartholomew ◽  
Raffaele De Francesco ◽  
Giacomo Paonessa
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Line ◽  
Genome Structure ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Naturally Occurring

ABSTRACT Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

scholarly journals Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 867
Author(s):  
Olga Povolyaeva ◽  
Yaroslava Chalenko ◽  
Egor Kalinin ◽  
Olga Kolbasova ◽  
Elena Pivova ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Domestic Animals ◽  
Epithelial Cell Line ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Listeria Monocytogenes Infection ◽  
Natural Hosts

L. monocytogenes is a widespread facultative intracellular pathogen. The range of natural hosts that supporting L. monocytogenes persistence in the environment has not been fully established yet. In this study, we were interested in the potential of L. monocytogenes to infect cells of bats, which are being increasingly recognized as a reservoir for microorganisms that are pathogenic to humans and domestic animals. A stable epithelial cell line was developed from the kidneys of Pipistrellus nathusii, a small bat widely distributed across Europe. The wild-type L. monocytogenes strain EGDe infected this cell line with an invasion efficiency of 0.0078 ± 0.0009%. Once it entered bat cells, L. monocytogenes doubled within about 70 min. When L. monocytogenes lacked either of the major invasion factors, InlA and InlB, invasion efficiency decreased by a factor of 10 and 25 respectively (p < 0.000001). The obtained results suggest that bat epithelial cells are susceptible to L. monocytogenes infection and that L. monocytogenes invasion of bat cells depends on the major invasion factors InlA and InlB. These results constitute the first report on in vitro studies of L. monocytogenes infection in bats.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals Functional analysis of synovial fluid from osteoarthritic knee and carpometacarpal joints unravels different molecular profiles

Rheumatology ◽  
2018 ◽  
Vol 58 (5) ◽  
pp. 897-907 ◽  
Author(s):  
Goncalo Barreto ◽  
Rabah Soliymani ◽  
Marc Baumann ◽  
Eero Waris ◽  
Kari K Eklund ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Mrna Level ◽  
Digital Pcr ◽  
Label Free ◽  
Primary Chondrocytes ◽  
Functional Studies ◽  
Knee Oa ◽  
Carpometacarpal Joints

Abstract Objective In this work, we aimed to elucidate the molecular mechanisms driving primary OA. By studying the dynamics of protein expression in two different types of OA joints we searched for similarities and disparities to identify key molecular mechanisms driving OA. Methods For this purpose, human SF samples were obtained from CMC-I OA and knee joint of OA patients. SF samples were analysed by label-free quantitative liquid chromatography mass spectrometry. Disease-relevant proteins identified in proteomics studies, such as clusterin, paraoxonase/arylesterase 1 (PON1) and transthyretin were validated by enzyme-linked immunosorbent assays, and on the mRNA level by droplet digital PCR. Functional studies were performed in vitro using primary chondrocytes. Results Differential proteomic changes were observed in the concentration of 40 proteins including clusterin, PON1 and transthyretin. Immunoassay analyses of clusterin, PON1, transthyretin and other inflammatory cytokines confirmed significant differences in protein concentration in SF of CMC-I and knee OA patients, with primarily lower protein expression levels in CMC-I. Functional studies on chondrocytes unequivocally demonstrated that stimulation with SF obtained from knee OA, in contrast to CMC-I OA joint, caused a significant upregulation in pro-inflammatory response, cell death and hypertrophy. Conclusion This study demonstrates that differential expression of molecular players in SF from different OA joints evokes diverse effects on primary chondrocytes. The pathomolecular mechanisms of OA may significantly differ in various joints, a finding that brings a new dimension into the pathogenesis of primary OA.

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