Association of histone acetylation at the ACTA2 promoter region with epithelial mesenchymal transition of lens epithelial cells

Abstract

Purpose Epithelial mesenchymal transition (EMT) plays a central role in the development of fibrotic complications of the lens. The current study is designed to check whether EMT of lens epithelial cells (LECs) is regulated by epigenetic modifications and to evaluate the effect of Trichostatin-A (TSA) on the transforming growth factor-β (TGF-β)-induced EMT. Methods Fetal human LECs (FHL124) were treated with TGF-β2 in the presence or absence of TSA. Levels of mRNA, protein, as well as localization of α-smooth muscle actin (αSMA) were studied along with migration of LECs. Acetylation of histone H4 was analyzed and chromatin immunoprecipitation (ChIP) was carried out to study the level of acetylated histone H4 at the promoter of αSMA gene (ACTA2). Student's t-test was used for statistical analysis. Results TGF-β2 treatment resulted in myofibroblast-like changes and increased migratory capacity of FHL124. Protein and mRNA expression of αSMA increased, and immunofluorescence revealed presence of extensive stress fibers. TSA treatment preserved epithelial morphology, retarded cell migration, and abrogated an increase in αSMA levels. TSA led to the accumulation of acetylated histone H4 that was reduced on TGF-β2 treatment. However, increased level of histone H4 acetylation was found at the ACTA2 promoter region during TGF-β treatment.Conclusions The increased level of αSMA, a hallmark of EMT in LECs, is associated with increased level of histone H4 acetylation at its promoter region, and TSA helps in suppressing EMT by epigenetically reducing this level. TSA thus shows promising potential in management of fibrotic conditions of the lens.

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells

“…Fifteen short tandem repeat (STR) loci plus the gender determining locus, Amelogenin, were amplified using the PowerPlex® 16 HS System from Promega and processed using the ABI Prism® 3100-Avant Genetic Analyzer. Data analyzed using the GeneMapper® v4.0 Software from Applied Biosystems confirmed that the cell line was of a human male origin and showed a 96% match with the reported analysis [42] (Table 1). The cells were cultured in MEM (Life Technologies, Carlsbad, CA) containing 5% FBS (Life Technologies) and gentamycin/L-glutamate (Sigma-Aldrich, St. Louis, MO) and maintained in a humidified incubator at 37 °C containing 5% CO 2 .…”

Trichostatin a restores expression of adherens and tight junction proteins during transforming growth factor β-Mediated epithelial-to-mesenchymal transition

“…The figure shows phase contrast images of cells treated with 0.5 μM and 1 μM of TSA (labels on left) ±10 ng/ml TGF-β2 (labels above). [ 20 ]…”

“…The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the viability of LECs after TSA treatment. [ 20 ] Briefly, 7 × 10 3 cells per well were added to 96-well plates and incubated for 24 hours. The cells were treated with a wide range of TSA concentrations (0.1-5 μM) for 24 hours.…”

“…The detailed protocol has been reported previously. [ 20 ] Briefly, histones were crosslinked to DNA by incubating cells with 1% formaldehyde for ten min. Cells were then lysed and chromatin was sheared using a Branson SLPe150 sonifier (Branson Ultrasonics, Kowloon, Hong Kong).…”

“…[ 6 7 ] It has been previously shown that TSA helps in preventing EMT of the LECs by preserving its morphological features, preventing migration, and down-regulating the mesenchymal marker αSMA by modulating the acetylation status of its promoter. [ 20 ] However, the effect of TSA on epithelial markers such as E-cad, ZO-1, and β-cat, which are altered during EMT, has not been established. Also, information is lacking on the acetylation status of the promoter regions of these genes during EMT.…”