Dataset Information

Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway.

ABSTRACT:

Purpose

This study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers.

Methods

Human TM cells and TMSCs were exposed to tunicamycin, brefeldin A, or thapsigargin. Cell apoptosis was evaluated by flow cytometry. ER stress markers were detected by quantitative PCR, Western blotting, and immunostaining. Morphologic changes were evaluated by transmission electron microscopy. Cells were treated with the PERK inhibitor GSK2606414 or the elF2? dephosphorylation inhibitor Salubrinal together with tunicamycin to evaluate their effects on ER stress.

Results

Both TMSCs and TM cells underwent apoptosis after 48- and 72-hour treatment with ER stress inducers. ER stress triggered the unfolded protein response (UPR) with increased expression of GRP78, sXBP1, and CHOP, which was significantly lower in TMSCs than TM cells. Swollen ER and mitochondria were detected in both TMSCs and TM cells. Neither GSK2606414 nor salubrinal alone activated UPR. GSK2606414 significantly reduced cell survival rates after tunicamycin treatment, and salubrinal increased cell survival rates. The increased expression of GRP78, sXBP1, CHOP, and GADD34 peaked at 6 or 12 hours and lasted longer in TM cells than TMSCs. Salubrinal treatment dramatically increased OCT4 and CHI3L1 expression in TMSCs.

Conclusions

In response to ER stress inducers, TMSCs activated a lower level of UPR and lasted shorter than TM cells. Inhibition of elF2? dephosphorylation had a protective mechanism against cell death. Stem cells combined with salubrinal may be a more effective way for TM regeneration in glaucoma.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6340162 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:PurposeCells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.MethodsPrimary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment.ResultsInnate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions.ConclusionsWe demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.