scholarly journals Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

2010 ◽  
Vol 299 (3) ◽  
pp. G742-G750 ◽  
Author(s):  
P. Cong ◽  
V. Pricolo ◽  
P. Biancani ◽  
J. Behar
Keyword(s):  
Small Interfering Rna ◽  
Muscle Cells ◽  
High Cholesterol ◽  
Western Blots ◽  
Human Gallbladder ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Δ Opioid Receptor ◽  
Interfering Rna ◽  
Bulk Plasma

The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a δ-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.

scholarly journals Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 401-407
Author(s):  
Daniel Vocelle ◽  
Olivia M Chesniak ◽  
Milton R Smith ◽  
Christina Chan ◽  
S Patrick Walton
Keyword(s):  
Small Interfering Rna ◽  
Temporal Association ◽  
Delivery Vehicle ◽  
Intracellular Location ◽  
Recycling Endosomes ◽  
Intracellular Processing ◽  
Late Endosomes ◽  
Automated Microscopy ◽  
Interfering Rna ◽  
Early Late

Abstract Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.

Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts

2008 ◽  
Vol 411 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Veronica Lopez ◽  
Shannon L. Kelleher ◽  
Bo Lönnerdal
Keyword(s):  
Plasma Membrane ◽  
Cell Model ◽  
Matrix Metalloproteinase 2 ◽  
Cytotrophoblast Cell ◽  
Hypertonic Medium ◽  
Recycling Endosomes ◽  
Lactoferrin Receptor ◽  
Early Endosomes ◽  
Mammalian Tissues ◽  
Interfering Rna

LfR [Lf (lactoferrin) receptor] is expressed in most mammalian tissues, including placental trophoblasts, and is presumed to mediate the internalization of Lf. However, the physiological significance of trophoblast LfR is not understood. Using the CT (cytotrophoblast) cell model BeWo, we demonstrated that transfection with LfR siRNA (small interfering RNA) significantly decreased apo- but not holo-Lf uptake compared with mock-transfected controls and that apo- but not holo-Lf significantly increased MMP (matrix metalloproteinase)-2 activity. As Lf functionality is related to the presence (holo-Lf) or absence (apo-Lf) of iron within the Lf molecule, our results suggest that apo-Lf may play a role in cellular invasion. Moreover, we detected LfR (∼105 kDa) in association with the plasma membrane, and ligand blotting confirmed that Lf binds to a LfR of ∼105 kDa. Apo-Lf treatment significantly increased LfR abundance at the plasma membrane and internalization probably occurs via clathrin-mediated endocytosis through early and recycling endosomes, as LfR was co-localized with EEA1 (early endosome antigen 1) and TfR (transferrin receptor) using confocal microscopy, and hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. In summary, our data demonstrate that apo- but not holo-Lf is internalized by LfR and suggest that, following internalization via LfR, apo-Lf plays a role in CT invasiveness by inducing MMP-2 activity. Moreover, LfR facilitates apo-Lf uptake specifically through clathrin-mediated endocytosis into early endosomes and potentially into a recycling pathway. Taken together, our data provide a new dimension in understanding ligand-dependant function that may be directly related to the ability of LfR to selectively internalize apo- but not holo-Lf.

scholarly journals Mechanisms regulating targeting of recycling endosomes to the cleavage furrow during cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 391-394 ◽  
Author(s):  
Glenn C. Simon ◽  
Rytis Prekeris
Keyword(s):  
Small Interfering Rna ◽  
Cleavage Furrow ◽  
Gtpase Activating Protein ◽  
Successful Completion ◽  
Interacting Protein ◽  
Recycling Endosomes ◽  
Spatial Regulation ◽  
Dividing Cells ◽  
Temporal And Spatial ◽  
Interfering Rna

Recently, recycling endosomes have emerged as a key components required for the successful completion of cytokinesis. Furthermore, FIP3 (family of Rab11-interacting protein 3), a Rab11 GTPase-binding protein, has been implicated in targeting the recycling endosomes to the midbody of dividing cells. Previously, we have shown that FIP3/Rab11-containing endosomes associate with centrosomes until anaphase, at which time they translocate to the cleavage furrow. At telophase, FIP3/Rab11-containing endosomes move from the furrow into the midbody, and this step is required for abscission. While several other proteins were implicated in regulating FIP3 targeting to the cleavage furrow, the mechanisms regulating the dynamics of FIP3-containing endosomes during mitosis have not been defined. To identify the factors regulating FIP3 targeting to the furrow, we used a combination of siRNA (small interfering RNA) screens and proteomic analysis to identify Cyk-4/MgcRacGAP (GTPase-activating protein) and kinesin I as FIP3-binding proteins. Furthermore, kinesin I mediates the transport of FIP3-containing endosomes to the cleavage furrow. Once in the furrow, FIP3 binds to Cyk-4 as part of centralspindlin complex and accumulates at the midbody. Finally, we demonstrated that ECT2 regulates FIP3 association with the centralspindlin complex. Thus we propose that kinesin I, in concert with centralspindlin complex, plays a role in temporal and spatial regulation of endosome transport to the cleavage furrow during cytokinesis.

Depletion of β-COP reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1

2008 ◽  
Vol 294 (6) ◽  
pp. C1485-C1498 ◽  
Author(s):  
Melanie L. Styers ◽  
Amber K. O'Connor ◽  
Robert Grabski ◽  
Estelle Cormet-Boyaka ◽  
Elizabeth Sztul
Keyword(s):  
Spatial Differentiation ◽  
Structural Protein ◽  
Recycling Endosomes ◽  
Protein Traffic ◽  
Caveolin 1 ◽  
Early Endosomes ◽  
Traffic Reduction ◽  
The Common ◽  
Golgi Network ◽  
Interfering Rna

We have utilized small interfering RNA (siRNA)-mediated depletion of the β-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in β-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the γ-subunit of COP-I, suggesting that Cav1 is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1.

STAT3-Driven, Aberrantly Expressed Lipoprotein Lipase Mediates Free Fatty Acid Metabolism in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3304-3304
Author(s):  
Uri Rozovski ◽  
Srdana Grgurevic ◽  
Carlos E. Bueso-Ramos ◽  
David M. Harris ◽  
Ping Li ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lipoprotein Lipase ◽  
Small Interfering Rna ◽  
Research Funding ◽  
Muscle Cells ◽  
Lymphocytic Leukemia ◽  
Proliferation Rate ◽  
Luciferase Reporter ◽  
Interfering Rna

Abstract The metabolic profile of mammalian cells is determined primarily by the cells’ proliferation rate. Unlike circulating memory B cells, which are typically quiescent and proliferate only in response to external stimuli, approximately 1% of chronic lymphocytic leukemia (CLL) cells proliferate daily. We sought to determine how CLL cells adjust their metabolism to meet increased energy demands imposed by their proliferation rate. Muscle cells, which proliferate at rates similar to those of CLL cells, preferentially use intracellular stored triglycerides as an available energy source. Similar to muscle cells, CLL cells express lipoprotein lipase (LPL), an enzyme that catalyzes the hydrolysis of tryglycerides into free fatty acids (FFA). We wondered whether CLL cells use a similar pathway. In reviewing bone marrow biopsies of patients with CLL, we identified clear-appearing oil red O-positive vacuoles in the cytoplasm of CLL cells. Using electron microscopy, we confirmed that these lipid vacuoles were present in 95% of CLL peripheral blood cells but not in normal B cells. To determine whether CLL cells metabolize FFA, we incubated CLL cells with or without FFA (palmitate or oleate) in a sealed flask and measured the dissolved O2 (dO2) content in the medium of the cultured cells after 48 h. Compared with CLL cells incubated in the absence of FFA, dO2 levels were significantly reduced when FFA was added. In contrast, dO2 levels were not reduced after FFA was added to cultures of normal B lymphocytes, suggesting that unlike normal B cells, CLL cells acquired the capacity to metabolize FFA. Transfecting CLL cells with LPL small interfering RNA abrogated the capacity of CLL cells to metabolize FFA, suggesting that FFA metabolism in CLL cells is LPL dependent. We and other groups found that LPL is abundantly expressed in CLL cells. Because STAT3 is constitutively activated in CLL cells and because we identified putative STAT3 binding sites in the LPL promoter, we hypothesized that STAT3 induces aberrant expression of LPL in CLL cells. By transfecting a luciferase reporter gene driven by LPL promoter fragments into MM1 cells, we found that STAT3 activates the LPL promoter, and by using chromatin immunoprecipitation and electrophoretic mobility shift assays, we confirmed that STAT3 binds to the LPL promoter in MM1 and in CLL cells. To confirm these data, we transfected CLL cells with a lentiviral STAT3 short hairpin RNA. Unlike the empty lentiviral vector, STAT3–small interfering RNA downregulated mRNA levels of LPL and several STAT3 target genes and downregulated LPL protein levels. Taken together, our data suggest that CLL cells store lipids in cytoplasmic vacuoles, produce LPL, and adapt their metabolism to utilize intracellular stored lipids for energy production, a process that is driven by constitutively activated STAT3. Disclosures O'Brien: Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding.

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries

Vascular ◽  
2013 ◽  
Vol 21 (5) ◽  
pp. 301-306 ◽  
Author(s):  
He Xia ◽  
Ji Jun ◽  
Ling Wen-ping ◽  
Pan Yi-feng ◽  
Chen Xiao-ling
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Iliac Artery ◽  
Small Interfering Rna ◽  
Muscle Cells ◽  
Platelet Derived Growth Factor ◽  
Factor B ◽  
Injury Model ◽  
Chitosan Nanoparticle ◽  
Interfering Rna

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model.

scholarly journals Coculture with Primary Visceral Rat Adipocytes from Control But Not Streptozotocin-Induced Diabetic Animals Increases Glucose Uptake in Rat Skeletal Muscle Cells: Role of Adiponectin

Endocrinology ◽  
2007 ◽  
Vol 148 (9) ◽  
pp. 4411-4419 ◽  
Author(s):  
Vivian Vu ◽  
Wi Kim ◽  
Xiangping Fang ◽  
Yuan-Tao Liu ◽  
Aimin Xu ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Glucose Uptake ◽  
High Molecular Weight ◽  
Small Interfering Rna ◽  
Muscle Cells ◽  
Rat Skeletal Muscle ◽  
Rat Adipocytes ◽  
Adiponectin Receptor 2 ◽  
Interfering Rna

We developed a coculture system comprising primary rat adipocytes and L6 rat skeletal muscle cells to allow investigation of the effects of physiologically relevant mixtures of adipokines. We observed that coculture, or adipocyte-conditioned media, increased glucose uptake in muscle cells. An adipokine that could potentially mediate this effect is adiponectin, and we demonstrated that small interfering RNA-mediated knockdown of adiponectin receptor-2 in muscle cells reduced the uptake of glucose upon coculture with primary rat adipocytes. Analysis of coculture media by ELISA indicated total adiponectin concentration of up to 1 μg/ml, and Western blotting and gel filtration analysis demonstrated that the adipokine profile was hexamer greater than high molecular weight much greater than trimer. We used the streptozotocin-induced rat model of diabetes and found that high-molecular-weight adiponectin levels decreased in comparison with control animals and this correlated with the fact that diabetic rat-derived primary adipocytes in coculture did not stimulate glucose uptake to the same extent as control adipocytes. Coculture induced phosphorylation of AMP-activated protein kinase (T172) and interestingly also insulin receptor substrate-1 (Y612) and Akt (T308 & S473), which could be attenuated after adiponectin receptor-2-small interfering RNA treatment. In summary, we believe that this coculture system represents an excellent model to study the effects of primary adipocyte-derived adipokine mixtures on skeletal muscle metabolism, and here we have established that in the context of physiologically relevant mixtures of adipokines, adiponectin may be an important determinant of positive cross talk between adipocytes and skeletal muscle.

scholarly journals The Orphan Nuclear Receptor, NOR-1, Is a Target of β-Adrenergic Signaling in Skeletal Muscle

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5217-5227 ◽  
Author(s):  
Michael A. Pearen ◽  
James G. Ryall ◽  
Megan A. Maxwell ◽  
Naganari Ohkura ◽  
Gordon S. Lynch ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Muscle Mass ◽  
Nuclear Receptor ◽  
Cold Exposure ◽  
Small Interfering Rna ◽  
Muscle Cells ◽  
Orphan Nuclear Receptor ◽  
Expression Of Genes ◽  
Interfering Rna

β-Adrenergic receptor (β-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that β-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77−/− animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30–480 min of β-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific β2- and β3-AR agonists had similar effects as the pan-agonist and were blocked by the β-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to β-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.

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