Interleukin 9 induces expression of three cytokine signal inhibitors: cytokine-inducible SH2-containing protein, suppressor of cytokine signalling (SOCS)-2 and SOCS-3, but only SOCS-3 overexpression suppresses interleukin 9 signalling

2000 ◽  
Vol 353 (1) ◽  
pp. 109-116 ◽  
Author(s):  
Diane LEJEUNE ◽  
Jean-Baptiste DEMOULIN ◽  
Jean-Christophe RENAULD
Keyword(s):  
Signal Transduction ◽  
Cell Types ◽  
Janus Kinase ◽  
Signal Attenuation ◽  
Hek 293 ◽  
293 Cells ◽  
Apoptotic Activity ◽  
Induced Signal ◽  
Interleukin 9

Interleukin 9 (IL-9) is a cytokine preferentially produced by T helper type 2 lymphocytes and active on various cell types such as T- and B-lymphocytes, mast cells and haemopoietic progenitors. The IL-9 receptor (IL-9R) belongs to the haemopoietic receptor superfamily and its signal transduction involves mainly the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Here we studied the implication of a novel family of suppressors of cytokine signalling (called CIS, for cytokine-inducible SH2-containing protein, and SOCS, for suppressor of cytokine signalling) in IL-9 signal attenuation. In BW5147 T-cell lymphoma, IL-9 induced the rapid expression of CIS, SOCS-2 and SOCS-3 with a peak after 2h of stimulation. Using IL-9R mutants, we showed that STAT activation is required for CIS/SOCS induction: CIS and SOCS-2 expression was induced either via STAT1 and/or STAT3 or via STAT5 but only STAT1 and/or STAT3 were involved in SOCS-3 expression. The effect of these three proteins on IL-9 signal transduction was assessed by transient transfection in HEK-293 cells expressing the components of the IL-9 signalling pathway and a STAT-responsive reporter construct. These experiments showed that only SOCS-3 is able to inhibit IL-9-induced signal transduction; neither CIS nor SOCS-2 exerted any effect. Stable transfection of CIS and SOCS-3 in BW5147 lymphoma cells showed that only overexpression of SOCS-3 had an inhibitory activity on STAT activation, gene induction and the anti-apoptotic activity of IL-9. By contrast, CIS failed to affect the IL-9 response.

scholarly journals The E3 Ubiquitin Ligase TEB4 Mediates Degradation of Type 2 Iodothyronine Deiodinase

2009 ◽  
Vol 29 (19) ◽  
pp. 5339-5347 ◽  
Author(s):  
Ann Marie Zavacki ◽  
Rafael Arrojo e Drigo ◽  
Beatriz C. G. Freitas ◽  
Mirra Chung ◽  
John W. Harney ◽  
...  
Keyword(s):  
Cell Types ◽  
Iodothyronine Deiodinase ◽  
Hek 293 ◽  
Protein Levels ◽  
Hematopoietic Lineage ◽  
293 Cells ◽  
Brown Adipose ◽  
Critical Instability ◽  
Proteasomal Inhibitor

ABSTRACT The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by ∼50% (P < 0.05). A highly efficient TEB4 knockdown (>90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Δ18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 μM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.

scholarly journals Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hongfei Chen ◽  
Zhousheng Jin ◽  
Fangfang Xia ◽  
Zhijian Fu
Keyword(s):  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation

2009 ◽  
Vol 297 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Ben J. Gu ◽  
Catherine Rathsam ◽  
Leanne Stokes ◽  
Andrew B. McGeachie ◽  
James S. Wiley
Keyword(s):  
Specific Inhibitor ◽  
Life Time ◽  
Cell Types ◽  
Interferon Γ ◽  
Extracellular Atp ◽  
Nonmuscle Myosin ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Myosin Va

The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase

2013 ◽  
Vol 394 (5) ◽  
pp. 685-693 ◽  
Author(s):  
Petra Cordes ◽  
Wei Sun ◽  
Rainer Wolber ◽  
Ludger Kolbe ◽  
Gerhard Klebe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heterologous Expression ◽  
Western Blotting ◽  
Kluyveromyces Lactis ◽  
Cell Types ◽  
Hek 293 ◽  
Activity Staining ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Human Tyrosinase

Abstract Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

scholarly journals Bupivacaine inhibits a small-conductance calcium-activated potassium type 2 channel (SK2) in HEK293 cells

2020 ◽  
Author(s):  
Hongfei Chen ◽  
Fangfang Xia ◽  
Zhousheng Jin ◽  
Zhijian Fu
Keyword(s):  
Hek293 Cells ◽  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause severe cardiotoxicity. Small conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unknown. This study investigated the effect of bupivacaine on SK2 channels.Methods: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch clamp technique was used to study the effect of bupivacaine on SK2 channels. Concentration-response relationship of bupivacaine for inhibiting SK2 current (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined.Results: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine and lidocaine on the SK2 current was 16.5, 46.5, and 77.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium.Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity.

scholarly journals Maitotoxin converts the plasmalemmal Ca2+ pump into a Ca2+-permeable nonselective cation channel

2009 ◽  
Vol 297 (6) ◽  
pp. C1533-C1543 ◽  
Author(s):  
William G. Sinkins ◽  
Mark Estacion ◽  
Vikram Prasad ◽  
Monu Goel ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Cell Types ◽  
Cation Channel ◽  
Nonselective Cation Channel ◽  
Whole Cell ◽  
Hek 293 ◽  
Marine Toxin ◽  
Hek Cells ◽  
293 Cells ◽  
Enhanced Expression ◽  
Interfering Rna

Maitotoxin (MTX) activates Ca2+-permeable nonselective cation channels and causes a dramatic increase in cytosolic free Ca2+ concentration ([Ca2+]i) in every cell examined to date, but the molecular identity of the channels involved remains unknown. A clue came from studies of a structurally related marine toxin called palytoxin (PTX). PTX binds to the plasmalemmal Na+-K+-ATPase (NKA) and converts the Na+ pump into a nonselective cation channel. Given the high permeability of the MTX channel for Ca2+, we considered the possibility that MTX may bind to the plasmalemmal Ca2+-ATPase (PMCA) pump, and like PTX, convert the pump into a channel. To test this hypothesis, the PMCA was overexpressed in Spodoptera frugiperda (Sf9) insect cells and in human embryonic kidneys (HEK) 293 cells. In both cell types, enhanced expression of the PMCA was associated with a significant increase in MTX-induced whole cell membrane currents. The effect of MTX on whole cell currents in both wild-type and PMCA overexpressing HEK cells was sensitive to pump ligands including Ca2+ and ATP. MTX-induced currents were significantly reduced by knockdown of PMCA1 in HEK cells using small interfering RNA or in mouse embryonic fibroblasts from genetically modified mice with the PMCA1(+/−) PMCA4(−/−) genotype. Finally, PMCA catalytic activity (i.e., Ca2+-ATPase) in isolated membranes, or in purified PMCA preparations, was inhibited by MTX. Together, these results suggest that MTX binds to and converts the PMCA pump into a Ca2+-permeable nonselective cation channel.

Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases

2003 ◽  
Vol 284 (2) ◽  
pp. F293-F302 ◽  
Author(s):  
Ibrahim Çetinkaya ◽  
Giuliano Ciarimboli ◽  
Gülay Yalçinkaya ◽  
Thomas Mehrens ◽  
Ana Velic ◽  
...  
Keyword(s):  
Myosin Light Chain ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Substrate Affinity ◽  
Phosphatidylinositol 3 Kinase ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Signaling Pathway ◽  
Initial Uptake

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]- N-methylpyridinium (ASP+). ASP+uptake was electrogenic and inhibited by TPA+(EC50= 2.7 μM), tetraethylammonium (EC50= 35 μM), cimetidine (EC50= 36 μM), or quinine (EC50= 6.7 μM). Stimulation with carbachol or ATP decreased initial uptake by 44 ± 3 ( n = 14) and 34 ± 4% ( n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 ± 4% ( n = 40). Inhibition of calmodulin (CaM), Ca2+/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 ± 2 ( n = 15), 40 ± 4 ( n = 30), and 31 ± 4% ( n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC50value for the inhibition of ASP+uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.

Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3

2009 ◽  
Vol 296 (5) ◽  
pp. C1105-C1114 ◽  
Author(s):  
P. Charukeshi Chandrasekera ◽  
Margaret E. Kargacin ◽  
Julie P. Deans ◽  
Jonathan Lytton
Keyword(s):  
Cell Types ◽  
Protein Isoforms ◽  
Inhibitory Antibody ◽  
Human Tonsil ◽  
Hek 293 ◽  
293 Cells ◽  
Plasmic Reticulum ◽  
Two Component ◽  
Calcium Affinity ◽  
First Time

The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca2+ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca2+; however, Ca2+ affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca2+ uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca2+ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca2+ affinity for SERCA3 compared with SERCA2b (1.10 ± 0.04 vs. 0.26 ± 0.01 μM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca2+ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca2+ affinity in these two preparations was 1.04 ± 0.07 and 1.1 ± 0.2 μM for SERCA3 and 0.27 ± 0.02 and 0.26 ± 0.01 μM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca2+ is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.

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