Insulin and growth hormone stimulate somatostatin receptor (SSTR) expression by inducing transcription of SSTR mRNAs and by upregulating cell surface SSTRs

2006 ◽  
Vol 291 (1) ◽  
pp. R163-R169 ◽  
Author(s):  
Laura E. Nelson ◽  
Mark A. Sheridan
Keyword(s):  
Growth Hormone ◽  
Rainbow Trout ◽  
Cell Surface ◽  
Somatostatin Receptor ◽  
Somatostatin Receptors ◽  
Chinese Hamster ◽  
Functional Expression ◽  
Surface Expression ◽  
Dependent Manner ◽  
The Stability

This study examined the effects of insulin (INS) and growth hormone (GH) on mRNA and functional expression of somatostatin receptors (SSTRs). Rainbow trout liver was used as a model system to evaluate the direct effects of INS and GH on mRNA expression of three SSTR subtypes characterized previously from this species: SSTR1A, SSTR1B, and SSTR2. INS and GH directly stimulated steady-state levels of all SSTR mRNAs in a concentration- and time-dependent manner; however, the pattern of expression was hormone and SSTR subtype specific. INS stimulated SSTR2 expression to a greater extent than SSTR1A or SSTR1B expression, whereas GH stimulated SSTR2 and SSTR1B expression to a similar extent, with SSTR2 and SSTR1B expression being more responsive to GH than SSTR1A. Whether INS- or GH-stimulated SSTR expression resulted from altered rates of transcription and/or changes in mRNA stability also was investigated. Formation of nascent SSTR transcripts in nuclei isolated from rainbow trout hepatocytes was significantly stimulated by INS and GH. Neither INS nor GH, however, affected the stability of SSTR mRNAs. Functional expression of SSTRs was studied in Chinese hamster ovary (CHO-K1) cells stably transfected with SSTR1A or SSTR1B. Surface expression of functional SSTRs was stimulated by INS and GH. These findings indicate that INS and GH stimulate SSTR expression by regulating transcription of SSTR mRNAs and by increasing functional SSTRs on the cell surface, and they suggest that regulation of SSTRs may be important for the coordination of growth, development, and metabolism of vertebrates.

scholarly journals Differential ligand binding and agonist-induced regulation characteristics of the two rainbow trout GH receptors, Ghr1 and Ghr2, in transfected cells

2009 ◽  
Vol 202 (3) ◽  
pp. 463-471 ◽  
Author(s):  
Katie M Reindl ◽  
Jeffrey D Kittilson ◽  
Mark A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Cell Binding ◽  
Single Class ◽  
Transfected Cells ◽  
Target Organs

Previously, we isolated and characterized two distinct GH receptor (GHR)-encoding mRNAs, ghr1 and ghr2, from rainbow trout. In this study, Chinese hamster ovary-K1 cells were individually transfected with plasmids that contained cDNAs encoding rainbow trout ghr1 or ghr2. High affinity binding of 125I-salmonid GH (sGH) by the expressed receptors was saturable, displaceable, and ligand selective. Whole-cell binding analysis revealed a single class of binding site; for Ghr1 Kd=8 nM, for Ghr2 Kd=17 nM. While salmonid prolactin (sPrl) displaced 125I-sGH from both Ghr1 and Ghr2, the affinity of either receptor subtype for sPrl was substantially less than for sGH; salmonid somatolactin, another member of the GH–PRL family, did not displace labeled sGH except at pharmacological concentrations. 125I-sGH was internalized by Ghr1- and Ghr2-expressing cells in a time-dependent manner; the maximum internalization reached was 71% for Ghr1 and 55% for Ghr2. Long-term exposure (24 h) of transfected cells to sGH up-regulated surface expression of both Ghr1 and Ghr2; however, sGH induced surface expression of Ghr1 to a greater extent than that of Ghr2. These results indicate that rainbow trout ghrs display both overlapping and distinct characteristics that may be important for ligand selection and differential action in target organs.

scholarly journals Somatostatin receptors subtypes 2 and 5, dopamine receptor type 2 expression and gsp status as predictors of octreotide LAR® responsiveness in acromegaly

2008 ◽  
Vol 52 (8) ◽  
pp. 1288-1295 ◽  
Author(s):  
Leonardo Vieira Neto ◽  
Giselle Fernandes Taboada ◽  
Mônica Roberto Gadelha
Keyword(s):  
Growth Hormone ◽  
Quantitative Analysis ◽  
Dopamine Receptor ◽  
Molecular Analysis ◽  
Somatostatin Receptor ◽  
Somatostatin Receptors ◽  
Laboratory Data ◽  
Octreotide Lar ◽  
Acute Test ◽  
Igf I

We present two acromegalic patients in which clinical and molecular data are discussed in regard to their ability to predict long term octreotide LAR® therapy response. Case reports: Patient 1: female, 36 years old at diagnosis. Basal GH and IGF-I at diagnosis were 133 ng/mL and 181% above the upper limit of reference values (ULRV), respectively. Growth hormone during acute test with subcutaneous octreotide decreased from 133 to 13 ng/mL. Patient started on primary octreotide LAR® therapy (20mg q28 days) and achieved biochemical parameters of disease control after 6 months. Molecular analysis of tumor fragments: gsp +; quantitative analysis of SSTR (somatostatin receptor) and DR (dopamine receptor) mRNA - SSTR2 23954; SSTR5 2407; DR2 total 17016 copies. Patient 2: male, 38 years old at diagnosis. Basal GH and IGF-I at diagnosis were 120 ng/mL and 114% ULRV, respectively. Patient underwent non-curative trans-sphenoidal surgery. Post-operative GH and IGF-I were 112 ng/mL and 137% ULRV, respectively. Growth hormone during acute test with subcutaneous octreotide decreased from 112 to 7 ng/mL. Octreotide LAR® therapy (20 mg q28 days) was then initiated. After 6 months of treatment, patient did not attain biochemical control of disease and displayed increased tumor volume. Molecular analysis of tumor fragments: gsp not done; quantitative analysis of SSTR and DR mRNA - SSTR2 416; SSTR5 3767; DR2 total 3439 copies. In conclusion, these two cases illustrate how laboratory data can be conflicting as predictors of octreotide LAR® responsiveness and how molecular analysis of tumor fragments can help explain different behaviors in clinically similar patients.

scholarly journals Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

2013 ◽  
Vol 304 (1) ◽  
pp. H94-H103 ◽  
Author(s):  
Aude Belliard ◽  
Yoann Sottejeau ◽  
Qiming Duan ◽  
Jessa L. Karabin ◽  
Sandrine V. Pierre
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Ion Transport ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Resistant Mutant ◽  
Surface Expression ◽  
Live Cells ◽  
Mutant Form ◽  
Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

scholarly journals Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

2021 ◽  
Vol 22 (19) ◽  
pp. 10207
Author(s):  
Julien Vitry ◽  
Guillaume Paré ◽  
Andréa Murru ◽  
Xavier Charest-Morin ◽  
Halim Maaroufi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Activation ◽  
Secretory Pathway ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Inhibitory Receptor ◽  
Lectin Receptors ◽  
Stalk Domain

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

scholarly journals Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana R. V. Pedro ◽  
Tânia Lima ◽  
Ricardo Fróis-Martins ◽  
Bárbara Leal ◽  
Isabel C. Ramos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Feed Supplements ◽  
Surface Receptor ◽  
Dose Dependent ◽  
Pro Inflammatory Cytokines

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

2001 ◽  
Vol 358 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Philippe ULSEMER ◽  
Catherine STRASSEL ◽  
Marie-Jeanne BAAS ◽  
Jean SALAMERO ◽  
Sylvette CHASSEROT-GOLAZ ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Ovary Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Vessel Injury ◽  
Von Willebrand

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

scholarly journals All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Thi Xoan Hoang ◽  
Jong Hyeok Jung ◽  
Jae Young Kim
Keyword(s):  
Retinoic Acid ◽  
Cell Surface ◽  
Human Monocyte ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Proinflammatory Responses

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1β in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1β production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis

1999 ◽  
Vol 339 (1) ◽  
pp. 185-192 ◽  
Author(s):  
Reika WATANABE ◽  
Kazuhito OHISHI ◽  
Yusuke MAEDA ◽  
Nobuo NAKAMURA ◽  
Taroh KINOSHITA
Keyword(s):  
Cell Surface ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Amino Acid Identity ◽  
Eukaryotic Cell ◽  
Surface Proteins ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Expression Cloning

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

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