scholarly journals Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts

2004 ◽  
Vol 15 (7) ◽  
pp. 3132-3145 ◽  
Author(s):  
Eeva-Liisa Eskelinen ◽  
Christine Katrin Schmidt ◽  
Silja Neu ◽  
Marion Willenborg ◽  
Graciela Fuertes ◽  
...  
Keyword(s):  
Fibroblast Cell ◽  
Cholesterol Homeostasis ◽  
Receptor Expression ◽  
Autophagic Vacuoles ◽  
Lysosome Biogenesis ◽  
Degradation Rates ◽  
Percoll Gradients ◽  
Chaperone Mediated Autophagy ◽  
Fibroblast Cell Lines

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.

scholarly journals Role of LAMP-2 in Lysosome Biogenesis and Autophagy

2002 ◽  
Vol 13 (9) ◽  
pp. 3355-3368 ◽  
Author(s):  
Eeva-Liisa Eskelinen ◽  
Anna Lena Illert ◽  
Yoshitaka Tanaka ◽  
Günter Schwarzmann ◽  
Judith Blanz ◽  
...  
Keyword(s):  
Half Life ◽  
Lysosomal Enzymes ◽  
State Level ◽  
Autophagic Vacuoles ◽  
Lysosome Biogenesis ◽  
Golgi Region ◽  
Activity Measurements ◽  
Phenotype Analysis ◽  
Mannose 6 Phosphate

In LAMP-2–deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2–deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2–deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.

scholarly journals Attenuation of glucocorticoid functions in an Anx-A1-/- cell line

2003 ◽  
Vol 371 (3) ◽  
pp. 927-935 ◽  
Author(s):  
Jamie D. CROXTALL ◽  
Derek W. GILROY ◽  
Egle SOLITO ◽  
Qamrul CHOUDHURY ◽  
Barbara J. WARD ◽  
...  
Keyword(s):  
Calf Serum ◽  
Fibroblast Cell ◽  
Receptor Expression ◽  
Cyclin Dependent Kinase ◽  
Extracellular Signal ◽  
Intracellular Organelles ◽  
Inflammatory Drugs ◽  
Cox 2 ◽  
Cytosolic Pla2 ◽  
Fibroblast Cell Lines

The Ca2+- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A2 (PLA2) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1+/+ cells, lung fibroblast cell lines derived from the Anx-A1−/− mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1−/− cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA2 and secretory PLA2 and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10−8– 10−6 M) inhibition. Proliferation and serum-induced progression of Anx-A1+/+ cells from G0/G1 into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1−/− cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1−/− cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.

scholarly journals The role of oxidative stress in 63 T-induced cytotoxicity against human lung cancer and normal lung fibroblast cell lines

2018 ◽  
Vol 37 (5) ◽  
pp. 849-864 ◽  
Author(s):  
Malgorzata Kucinska ◽  
Helena Mieszczak ◽  
Hanna Piotrowska-Kempisty ◽  
Mariusz Kaczmarek ◽  
Walter Granig ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lung Cancer ◽  
Cell Lines ◽  
Human Lung ◽  
Fibroblast Cell ◽  
Lung Fibroblast ◽  
Human Lung Cancer ◽  
Normal Lung ◽  
Fibroblast Cell Lines

scholarly journals Role of Cyclic mPer2 Expression in the Mammalian Cellular Clock

2005 ◽  
Vol 25 (5) ◽  
pp. 1912-1921 ◽  
Author(s):  
Yoshinobu Yamamoto ◽  
Kazuhiro Yagita ◽  
Hitoshi Okamura
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Constitutive Expression ◽  
Fibroblast Cell ◽  
Circadian Oscillation ◽  
Protein Accumulation ◽  
Oscillation System ◽  
Clock Gene Expression ◽  
Fibroblast Cell Lines

ABSTRACT To explore the role of mPer2 in the circadian oscillation in the mammalian cellular clock, we established fibroblast cell lines in which expression of mPer2 is controlled through a tetracycline-regulatable promoter. We revealed that constitutive expression and overexpression of mPer2 mRNA severely impair serum shock-induced cyclic circadian clock gene expression. Moreover, under conditions of lower mPer2 mRNA expression, mPER2 protein accumulation in these cells showed clear circadian oscillation even in constitutive mPer2 mRNA expression, suggesting that the protein cycling of mPER2 was required for oscillation of the circadian feedback loop. Since the rhythms of gene expression driven by the intrinsic clock oscillation system dampen rapidly in the absence of cyclic expression of mPer2, the transcriptional rhythm helps to sustain the clock oscillation.

scholarly journals Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.

1990 ◽  
Vol 1 (6) ◽  
pp. 471-486 ◽  
Author(s):  
F Verrey ◽  
T Gilbert ◽  
T Mellow ◽  
G Proulx ◽  
K Drickamer
Keyword(s):  
High Efficiency ◽  
Cytoplasmic Tail ◽  
Fibroblast Cell ◽  
Alternative Mechanism ◽  
Coated Pits ◽  
Chimeric Receptors ◽  
Receptor Localization ◽  
Cytoplasmic Tails ◽  
Fibroblast Cell Lines

Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism.

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

The role of hepatic 11[b]-hydroxysteroid dehydrogenase type 1 in cholesterol homeostasis

2013 ◽  
pp. 1-1
Author(s):  
Kajal Manwani ◽  
Tak Y Man ◽  
Christopher J Kenyon ◽  
Ruth Andrew ◽  
Karen E Chapman ◽  
...  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Cholesterol Homeostasis

scholarly journals Modulation of Macrophages M1/M2 Polarization Using Carbohydrate-Functionalized Polymeric Nanoparticles

Polymers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 88
Author(s):  
Raquel G. D. Andrade ◽  
Bruno Reis ◽  
Benjamin Costas ◽  
Sofia A. Costa Lima ◽  
Salette Reis
Keyword(s):  
Inflammatory Responses ◽  
Polymeric Nanoparticles ◽  
Interaction Mechanism ◽  
Mannose Receptor ◽  
The Body ◽  
Receptor Expression ◽  
Production Methods ◽  
Polymeric Nanocarriers ◽  
Efficient Delivery

Exploiting surface endocytosis receptors using carbohydrate-conjugated nanocarriers brings outstanding approaches to an efficient delivery towards a specific target. Macrophages are cells of innate immunity found throughout the body. Plasticity of macrophages is evidenced by alterations in phenotypic polarization in response to stimuli, and is associated with changes in effector molecules, receptor expression, and cytokine profile. M1-polarized macrophages are involved in pro-inflammatory responses while M2 macrophages are capable of anti-inflammatory response and tissue repair. Modulation of macrophages’ activation state is an effective approach for several disease therapies, mediated by carbohydrate-coated nanocarriers. In this review, polymeric nanocarriers targeting macrophages are described in terms of production methods and conjugation strategies, highlighting the role of mannose receptor in the polarization of macrophages, and targeting approaches for infectious diseases, cancer immunotherapy, and prevention. Translation of this nanomedicine approach still requires further elucidation of the interaction mechanism between nanocarriers and macrophages towards clinical applications.

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