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1.
Exp Cell Res ; 430(1): 113695, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37393981

ABSTRACT

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.


Subject(s)
GTP-Binding Proteins , Microscopy , Animals , Dogs , GTP-Binding Proteins/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Cell Division , Cell Proliferation , Mammals/metabolism
2.
Cell Signal ; 99: 110431, 2022 11.
Article in English | MEDLINE | ID: mdl-35933033

ABSTRACT

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.


Subject(s)
Adenocarcinoma , Pancreatic Neoplasms , Adenocarcinoma/metabolism , Cell Adhesion , Cell Proliferation , Epithelial Cells/metabolism , Humans , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase Kinases/metabolism , Pancreatic Neoplasms/metabolism , Phosphorylation , Signal Transduction , Transcription Factors/metabolism
3.
Biomolecules ; 10(8)2020 07 22.
Article in English | MEDLINE | ID: mdl-32707896

ABSTRACT

Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.


Subject(s)
Cell Movement/physiology , Cell Polarity/physiology , Fibroblasts/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Animals , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Line , Cell Movement/genetics , Cell Polarity/genetics , Cell Shape/genetics , Cell Shape/physiology , Fibroblasts/cytology , Focal Adhesion Protein-Tyrosine Kinases/genetics , Microscopy, Phase-Contrast , RNA Interference , Rats , Receptors for Activated C Kinase/genetics , Receptors for Activated C Kinase/metabolism
4.
J Cell Mol Med ; 24(4): 2402-2415, 2020 02.
Article in English | MEDLINE | ID: mdl-31957261

ABSTRACT

Arthrospira platensis, a blue-green alga, is a popular nutraceutical substance having potent antioxidant properties with potential anti-carcinogenic activities. The aim of our study was to assess the possible anti-angiogenic effects of A platensis in an experimental model of pancreatic cancer. The effects of an A platensis extract were investigated on human pancreatic cancer cells (PA-TU-8902) and immortalized endothelial-like cells (Ea.hy926). PA-TU-8902 pancreatic tumours xenografted to athymic mice were also examined. In vitro migration and invasiveness assays were performed on the tested cells. Multiple angiogenic factors and signalling pathways were analysed in the epithelial, endothelial and cancer cells, and tumour tissue. The A platensis extract exerted inhibitory effects on both migration and invasion of pancreatic cancer as well as endothelial-like cells. Tumours of mice treated with A platensis exhibited much lesser degrees of vascularization as measured by CD31 immunostaining (P = .004). Surprisingly, the VEGF-A mRNA and protein expressions were up-regulated in pancreatic cancer cells. A platensis inhibited ERK activation upstream of Raf and suppressed the expression of ERK-regulated proteins. Treatment of pancreatic cancer with A platensis was associated with suppressive effects on migration and invasiveness with various anti-angiogenic features, which might account for the anticancer effects of this blue-green alga.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Neovascularization, Pathologic/drug therapy , Pancreatic Neoplasms/drug therapy , Spirulina/chemistry , Animals , Antioxidants/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Endothelial Cells/drug effects , Humans , Mice , Mice, Nude , Signal Transduction/drug effects , Up-Regulation/drug effects , Pancreatic Neoplasms
5.
Biochim Biophys Acta ; 1863(9): 2189-200, 2016 09.
Article in English | MEDLINE | ID: mdl-27212270

ABSTRACT

The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit.


Subject(s)
Fibroblasts/cytology , Fibroblasts/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , GTP-Binding Proteins/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/metabolism , Repressor Proteins/metabolism , Actins/metabolism , Animals , Cell Adhesion , Cell Movement , Cell Shape , Fibroblasts/enzymology , GTP-Binding Proteins/deficiency , Gene Knockdown Techniques , Gene Silencing , Models, Biological , Phenotype , Rats , Receptors for Activated C Kinase
6.
Cell Signal ; 25(12): 2743-51, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24012955

ABSTRACT

The ERK (extracellular signal-regulated kinases) cascade has an evolutionarily conserved three tier architecture consisting of protein kinases Raf, MEK (MAPK/ERK kinase) and ERK. Following activation, ERK phosphorylates various cellular elements leading to diverse cellular responses. Downstream of ERK the family of p90 ribosomal S6 kinases (RSKs) has been proven to be an important conveyor of ERK signaling, however, little is known if ERK and RSK coordinate their functions to generate a specific biological response. Here we show that in epithelial cells conditional activation of the ERK pathway causes phenotypic conversion of epithelial cells to autonomously migrating cells. This process involves two sequential steps characterized by loss of apical-basal polarity followed by cell scattering. The activation of ERK, but not RSK, is sufficient for the execution of the first step and it requires calpain mediated remodeling of actin cytoskeleton. Conversely, RSK regulates the successive stage characterized by cell-cell contact weakening and increased cellular migration. Thus, ERK and RSK regulate different cellular subprograms and coordinated execution of these subprograms in time generates a relevant biological response. Our data also suggest that the mechanism by which the ERK pathway controls a cellular response may be distributed between ERK and RSK, rather than being elicited by a single effector kinase.


Subject(s)
Epithelial Cells/cytology , Epithelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction , Animals , Cadherins/metabolism , Calpain/metabolism , Cell Line , Cell Movement , Cell Polarity , Dogs , Humans , Intercellular Junctions/metabolism , MAP Kinase Signaling System
7.
Food Chem Toxicol ; 59: 754-65, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23872132

ABSTRACT

Currently, there is a trend to reduce of parabens use due to concern about the safety of their unmetabolised forms. This paper focused on dermal absorption rate and effectiveness of first-pass biotransformation of methylparaben (MP) under in-use conditions of skincare products. 24-h exposure of previously frozen intact and tapestripped (20 strips) pig-ear skin to nine vehicles containing 0.1% MP (AD, applied dose of 10 µg/cm²), resulted in 2.0-5.8%AD and 2.9-7.6%AD of unmetabolised MP, and 37.0-73.0%AD and 56.0-95.0%AD of p-hydroxybenzoic acid, respectively, in the receptor fluid. The absorption rate of MP was higher from emulsions than from hydrogels, from enhancer-containing vehicles than from enhancer-free vehicles, and when skin was damaged. Experiments confirmed that the freezing of pig-ear skin slightly reduces hydrolysis of MP. After 4-h exposure of intact freshly excised and intact frozen stored skin, amount of

Subject(s)
Parabens/pharmacokinetics , Pharmaceutical Vehicles , Preservatives, Pharmaceutical/pharmacokinetics , Skin Absorption , Skin/injuries , Abattoirs , Administration, Cutaneous , Animals , Biotransformation , Cryopreservation , Dermatologic Agents/adverse effects , Dermatologic Agents/analysis , Ear , Emulsions , Female , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Male , Parabens/administration & dosage , Parabens/adverse effects , Parabens/analysis , Pharmaceutical Vehicles/chemistry , Preservatives, Pharmaceutical/administration & dosage , Preservatives, Pharmaceutical/adverse effects , Preservatives, Pharmaceutical/analysis , Skin/metabolism , Sus scrofa
9.
J Mol Biol ; 425(11): 2039-2055, 2013 Jun 12.
Article in English | MEDLINE | ID: mdl-23524135

ABSTRACT

The establishment of cell polarity is an essential step in the process of cell migration. This process requires precise spatiotemporal coordination of signaling pathways that in most cells create the typical asymmetrical profile of a polarized cell with nucleus located at the cell rear and the microtubule organizing center (MTOC) positioned between the nucleus and the leading edge. During cell polarization, nucleus rearward positioning promotes correct microtubule organizing center localization and thus the establishment of front-rear polarity and directional migration. We found that cell polarization and directional migration require also the reorientation of the nucleus. Nuclear reorientation is manifested as temporally restricted nuclear rotation that aligns the nuclear axis with the axis of cell migration. We also found that nuclear reorientation requires physical connection between the nucleus and cytoskeleton mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex. Nuclear reorientation is controlled by coordinated activity of lysophosphatidic acid (LPA)-mediated activation of GTPase Rho and the activation of integrin, FAK (focal adhesion kinase), Src, and p190RhoGAP signaling pathway. Integrin signaling is spatially induced at the leading edge as FAK and p190RhoGAP are predominantly activated or localized at this location. We suggest that integrin activation within lamellipodia defines cell front, and subsequent FAK, Src, and p190RhoGAP signaling represents the polarity signal that induces reorientation of the nucleus and thus promotes the establishment of front-rear polarity.


Subject(s)
Cell Movement , Cell Nucleus/metabolism , Cell Polarity , Fibroblasts/physiology , Animals , Cell Line , Cytoskeletal Proteins/metabolism , Fibroblasts/cytology , Rats , Signal Transduction
10.
Food Chem Toxicol ; 52: 19-27, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23127598

ABSTRACT

Currently, there is evidence of health risks of triphenylmethane dyes after systemic absorption. This paper investigates the fate of Brilliant Blue (BB) and Patent Blue (PB) after 24-h in vitro diffusion, firstly through intact and secondly through shaven pig-ear skin (stored by freezing) from four leave-on cosmetics under in-use conditions. Both dyes showed no measurable permeation through intact skin but significant permeation was found through shaven skin. From 250 ng/cm(2) of dye in one applied dose there were found 52 ng/cm(2) of BB and 91 ng/cm(2) of PB from ethanol-based after-shave, 39 ng/cm(2) of BB and 86 ng/cm(2) of PB from ethanol-free facial-cleanser, 35 ng/cm(2) of BB and 43 ng/cm(2) of PB from O/W emulsion, and no amount from W/O emulsion, as available to become systemically absorbed. Thirdly, the paper focuses on lingual mucosa after licking lollipops. Ex vivo porcine tongue dorsum was exposed to human saliva with 15,000 ng/cm(2) of dye for 20 min. 24-h diffusion resulted in 34 ng/cm(2) of BB and 86 ng/cm(2) of PB which can be directly absorbed into the blood system. Findings are troubling, particularly with regard to the frequent use of after-shave products by the male population and repeated lollipops licking by children.


Subject(s)
Benzenesulfonates/pharmacokinetics , Coloring Agents/pharmacokinetics , Skin Absorption , Trityl Compounds/pharmacokinetics , Animals , Cosmetics/pharmacokinetics , Diffusion , Emulsions/pharmacokinetics , Ethanol/pharmacokinetics , Hair Removal , Humans , Mouth Mucosa/drug effects , Mouthwashes/adverse effects , Mouthwashes/pharmacokinetics , Mucous Membrane/drug effects , Swine , Tongue/drug effects
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