Jinggang Yin

Quantitative AP-1 gene regulation by oxidative stress in the human retinal pigment epithelium

The purpose of this study was to characterize the early molecular responses to quantified levels of oxidative stress (OS) in the human retinal pigment epithelium (RPE). Confluent ARPE‐19 cells were cultured for 3 days in defined medium to stabilize gene expression. The cells were exposed to varying levels of OS (0–500 µM H2O2) for 1–8 h and gene expression was followed for up to 24‐h after OS. Using real‐time qPCR, we quantified the expression of immediate early genes from the AP‐1 transcription factor family and other genes involved in regulating the redox status of the cells. Significant and quantitative changes were seen in the expression of six AP‐1 transcription factor genes, FosB, c‐Fos, Fra‐1, c‐Jun, JunB, and ATF3 from 1–8 h following OS. The peak level of induced transcription from OS varied from 2‐ to 128‐fold over the first 4 h, depending on the gene and magnitude of OS. Increased transcription at higher levels of OS was also seen for up to 8‐h for some of these genes. Protein translation was examined for 24‐h following OS using Western blotting methods, and compared to the qPCR responses. We identified six AP‐1 family genes that demonstrate quantitative upregulation of expression in response to OS. Two distinct types of quantifiable OS‐specific responses were observed; dose‐dependent responses, and threshold responses. Our studies show that different levels of OS can regulate the expression of AP‐1 transcription factors quantitatively in the human RPE in vitro. J. Cell. Biochem. 108: 1280–1291, 2009.

Modulation of oxidative stress responses in the human retinal pigment epithelium following treatment with vitamin C.

Oxidative stress (OS) in the retina plays an important role in the development and progression of age‐related macular degeneration (AMD). Our previous work has shown that OS can quantitatively regulate the expression of AP‐1 family genes in the retinal pigment epithelium (RPE). In this study, we sought to determine whether AP‐1 genes can be used as cellular biomarkers of OS to evaluate the efficacy of ascorbate, the major aqueous‐phase antioxidant in the blood, in reducing OS in RPE cells in vitro. Human ARPE19 cells were pretreated with increasing levels of ascorbate (0–500 µM) for 3 days which was then removed from the medium. OS was induced 24 h later by the addition of hydrogen peroxide for 1–4 h, to bring the final media concentration of H2O2 to 500 µM. FosB, c‐Fos, and ATF3 gene expression was examined from 0 to 24 h after OS. Pretreatment with 200 µM ascorbate maximally reduced the transcriptional OS response of AP‐1 genes by up to 87% after 1 and 4 h, compared to controls. One hundred micromolar of ascorbate provided a statistically significant, but far more modest effect. Ascorbate supplementation of 100–200 µM appears to strongly inhibit OS‐induced activation of AP‐1 in vitro, but pretreatment with higher levels of ascorbate conferred no additional advantage. These studies suggest that there are optimal levels of antioxidant supplementation to the RPE in vitro. Laboratory assays based upon transcription factor biomarkers may be useful to define beneficial molecular responses to new antioxidants, alternative dosing regimens, and to explore therapeutic efficacy in OS models in vitro. J. Cell. Physiol. 226: 2025–2032, 2011.

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

Prominin-1 Is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium

Purpose Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE). Methods Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins. Results Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function. Conclusions Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.

Knockdown of survivin results in inhibition of epithelial to mesenchymal transition in retinal pigment epithelial cells by attenuating the TGFβ pathway

Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.