scholarly journals Increase of calcium levels in epithelial cells induces translocation of calcium-binding proteins migration inhibitory factor-related protein 8 (MRP8) and MRP14 to keratin intermediate filaments

1995 ◽  
Vol 309 (2) ◽  
pp. 419-424 ◽  
Author(s):  
M Goebeler ◽  
J Roth ◽  
C van den Bos ◽  
G Ader ◽  
C Sorg
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Intermediate Filaments ◽  
Migration Inhibitory Factor ◽  
Binding Proteins ◽  
Inhibitory Factor ◽  
Ionophore A23187 ◽  
Related Protein ◽  
S 100 ◽  
Keratin Intermediate Filaments

Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca(2+)-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulphosuccinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca(2+)-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca(2+)-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca(2+)-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Interferon-gamma induces macrophage migration inhibitory factor synthesis and secretion by tubular epithelial cells

Nephrology ◽  
2003 ◽  
Vol 8 (3) ◽  
pp. 156-161 ◽  
Author(s):  
EDWINA K RICE ◽  
DAVID J NIKOLIC-PATERSON ◽  
PRUDENCE A HILL ◽  
CHRISTINE N METZ ◽  
RICHARD BUCALA ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Interferon Gamma ◽  
Migration Inhibitory Factor ◽  
Macrophage Migration Inhibitory Factor ◽  
Inhibitory Factor ◽  
Macrophage Migration ◽  
Tubular Epithelial Cells

scholarly journals Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells

2010 ◽  
Vol 298 (2) ◽  
pp. G233-G240 ◽  
Author(s):  
Jie Ma ◽  
Karen M. Harnett ◽  
Jose Behar ◽  
Piero Biancani ◽  
Weibiao Cao
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Transient Receptor Potential ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Transferase Activity ◽  
Ionophore A23187 ◽  
Acetyl Coa ◽  
Coa Transferase

Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA2) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA2, p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA2 by Western blot. Capsaicin induced phosphorylation of p38 and cPLA2. Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA2 phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA2. To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

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