Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

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Effects of ambient particulate matter on a reconstructed human corneal epithelium model

Scientific Reports ◽

10.1038/s41598-021-82971-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ryota Ko ◽

Masahiko Hayashi ◽

Miho Tanaka ◽

Tomoaki Okuda ◽

Chiharu Nishita-Hara ◽

...

Keyword(s):

Particulate Matter ◽

Cell Viability ◽

Corneal Epithelium ◽

Dependent Manner ◽

Ambient Particulate Matter ◽

Tissue Sections ◽

Human Corneal Epithelium ◽

Vitro Model ◽

Dose Dependent

AbstractWe evaluated the effects of ambient particulate matter (PM) on the corneal epithelium using a reconstructed human corneal epithelium (HCE) model. We collected two PM size fractions [aerodynamic diameter smaller than 2.4 µm: PM0.3–2.4 and larger than 2.4 µm: PM>2.4] and exposed these tissues to PM concentrations of 1, 10, and 100 µg/mL for 24 h. After exposure, cell viability and interleukin (IL) IL-6 and IL-8 levels were determined, and haematoxylin and eosin and immunofluorescence staining of the zonula occludens-1 (ZO-1) were performed on tissue sections. In addition, the effects of a certified reference material of urban aerosols (UA; 100 µg/mL) were also examined as a reference. The viability of cells exposed to 100 μg/mL UA and PM>2.4 decreased to 76.2% ± 7.4 and 75.4% ± 16.1, respectively, whereas PM0.3–2.4 exposure had a limited effect on cell viability. These particles did not increase IL-6 and IL-8 levels significantly even though cell viability was decreased in 100 μg/mL UA and PM>2.4. ZO-1 expression was reduced in a dose-dependent manner in all groups. Reconstructed HCE could be used as an in vitro model to study the effects of environmental PM exposure on ocular surface cell viability and inflammation.

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The neurorepellent, Slit2, prevents macrophage lipid loading by inhibiting CD36-dependent binding and internalization of oxidized low-density lipoprotein

Scientific Reports ◽

10.1038/s41598-021-83046-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Bushra Yusuf ◽

Ilya Mukovozov ◽

Sajedabanu Patel ◽

Yi-Wei Huang ◽

Guang Ying Liu ◽

...

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Super Resolution ◽

Low Density ◽

Dependent Manner ◽

Oxidized Low Density Lipoprotein ◽

Cortical Actin ◽

Cytoskeletal Remodeling ◽

Atherosclerotic Lesions ◽

Modified Lipoproteins

AbstractAtherosclerosis is characterized by retention of modified lipoproteins, especially oxidized low density lipoprotein (oxLDL) within the sub-endothelial space of affected blood vessels. Recruited monocyte-derived and tissue-resident macrophages subsequently ingest oxLDL by binding and internalizing oxLDL via scavenger receptors, particularly CD36. The secreted neurorepellent, Slit2, acting through its transmembrane receptor, Roundabout-1 (Robo-1), was previously shown to inhibit recruitment of monocytes into nascent atherosclerotic lesions. The effects of Slit2 on oxLDL uptake by macrophages have not been explored. We report here that Slit2 inhibits uptake of oxLDL by human and murine macrophages, and the resulting formation of foam cells, in a Rac1-dependent and CD36-dependent manner. Exposure of macrophages to Slit2 prevented binding of oxLDL to the surface of cells. Using super-resolution microscopy, we observed that exposure of macrophages to Slit2 induced profound cytoskeletal remodeling with formation of a thick ring of cortical actin within which clusters of CD36 could not aggregate, thereby attenuating binding of oxLDL to the surface of cells. By inhibiting recruitment of monocytes into early atherosclerotic lesions, and the subsequent binding and internalization of oxLDL by macrophages, Slit2 could represent a potent new tool to combat individual steps that collectively result in progression of atherosclerosis.

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Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3277 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3277-3279 ◽

Cited By ~ 4

Author(s):

G Tjaden ◽

A Aguanno ◽

R Kumar ◽

D Benincasa ◽

R M Gubits ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

G Protein ◽

Alpha Subunit ◽

Low Density ◽

Dependent Manner ◽

Nerve Growth ◽

Density Dependent ◽

Stimulatory G Protein ◽

Gs Alpha

Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment.

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Region-Specific Microtubule Transport in Motile Cells

The Journal of Cell Biology ◽

10.1083/jcb.151.5.1003 ◽

2000 ◽

Vol 151 (5) ◽

pp. 1003-1012 ◽

Cited By ~ 26

Author(s):

Anne-Marie C. Yvon ◽

Patricia Wadsworth

Keyword(s):

Growth Factor ◽

Cell Motility ◽

Cell Body ◽

Dependent Manner ◽

Retrograde Flow ◽

Cell Locomotion ◽

Quantitative Measurements ◽

Motile Cells ◽

Microtubule Transport ◽

Hepatocyte Growth

Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport depends on the dominant force in the region examined. MTs in the leading lamella move rearward relative to the substrate, as has been reported in newt cells (Waterman-Storer, C.M., and E.D. Salmon. 1997. J. Cell Biol. 139:417–434), whereas MTs in the cell body and in the retraction tail move forward, in the direction of cell locomotion. In the transition zone between the peripheral lamella and the cell body, a subset of MTs remains stationary with respect to the substrate, whereas neighboring MTs are transported either forward, with the cell body, or rearward, with actomyosin retrograde flow. In addition to transport, the photoactivated region frequently broadens, indicating that individual marked MTs are moved either at different rates or in different directions. Mark broadening is also observed in nonmotile cells, indicating that this aspect of transport is independent of cell locomotion. Quantitative measurements of the dissipation of photoactivated fluorescence show that, compared with MTs in control nonmotile cells, MT turnover is increased twofold in the lamella of HGF-treated cells but unchanged in the retraction tail, demonstrating that microtubule turnover is regionally regulated.

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Vitrigel-EIT (eye irritancy test) methods provide a brief assay system for estimating the changes of barrier function and architecture after exposing test chemicals to a human corneal epithelium model

Toxicology Letters ◽

10.1016/j.toxlet.2013.05.356 ◽

2013 ◽

Vol 221 ◽

pp. S165

Author(s):

Hiroyuki Yamaguchi ◽

Hajime Kojima ◽

Toshiaki Takezawa

Keyword(s):

Corneal Epithelium ◽

Barrier Function ◽

Test Methods ◽

Assay System ◽

Human Corneal Epithelium

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Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein

Biochemical Journal ◽

10.1042/bj3360381 ◽

1998 ◽

Vol 336 (2) ◽

pp. 381-386 ◽

Cited By ~ 60

Author(s):

Lothar GORETZKI ◽

Barbara M. MUELLER

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Receptor ◽

Dependent Manner ◽

Related Protein ◽

Gtp Binding ◽

Density Lipoprotein Receptor ◽

Dose Dependent ◽

Lipoprotein Receptor Related Protein

The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase–inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsα subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsα from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.

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