Primary Human Lens Epithelial Cells Research Articles

Transcriptome exploration of ferroptosis-related genes in TGFβ- induced lens epithelial to mesenchymal transition during posterior capsular opacification development

BackgroundPosterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFβ) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO.MethodsRNA sequencing (RNA-seq) was performed on both TGF-β2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsOur analysis revealed 1253 DEGs between TGF-β2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq.ConclusionsOur RNA-Seq data identified 38 ferroptosis-related genes in TGF-β2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-β2 stimulation.Graphical Our study initially observed ferroptosis related genes in primary human lens epithelial cells stimulated by TGF-β2. These findings may improve the understanding of the molecular mechanisms of PCO and provide a new direction for exploring the potential mechanisms of PCO

TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract.

Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.

Long Noncoding RNA NEAT1 Regulates TGF-β2-Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells through the miR-34a/Snail1 and miR-204/Zeb1 Pathways.

The aim of this study was to explore whether the long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1)/miR-34a/Snail1 and NEAT1/miR-204/Zeb1 pathways are involved in epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Primary human LECs (HLECs) were separated and cultured. Our results identified that TGF-β2 induces NEAT1 overexpression in a dose-dependent manner and a time-dependent manner. Additionally, TGF-β2 induced downregulation of E-cadherin and upregulation of fibronectin in primary HLECs through a NEAT1-dependent mechanism. Microarray analysis showed that NEAT1 overexpression inhibited the miR-34a and miR-204 levels in the LECs. The expression of miR-34a and miR-204 was decreased, and the levels of Snail1 and Zeb1 were elevated in human posterior capsule opacification- (PCO-) attached LECs and the LECs obtained from anterior subcapsular cataract (ASC) by quantitative RT-PCR (qRT-PCR). Mechanistic studies revealed that NEAT1 negatively regulates miR-34a or miR-204, and miR-34a or miR-204 directly targets Snail1 or Zeb1 by luciferase assay and RNA-binding protein immunoprecipitation assay, respectively. Overall, the NEAT1/miR-34a/Snail1 and NEAT1/miR-204/Zeb1 pathways are involved in TGF-β2-induced EMT of HLECs. In summary, TGF-β2 induces NEAT1 overexpression, which in turn suggests that NEAT1 acts as a ceRNA targeting Snail1 or Zeb1 by binding miR-34a or miR-204, and promotes the progression of EMT of LECs.

Long Noncoding RNA MALAT1 Acts as a Competing Endogenous RNA to Regulate TGF-β2 Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells by a MicroRNA-26a-Dependent Mechanism.

The aim of the present study was to characterize whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-26a/Smad4 axis is involved in epithelial–mesenchymal transition (EMT) of lens epithelial cells (LECs). Primary human LECs were separated and cultured. Microarray analysis showed that a total of 568 lncRNAs are differentially expressed in primary HLECs in the presence of TGF-β2 and MALAT1 is mostly significantly dysregulated lncRNAs, which is increased by nearly 17-fold. In addition, upregulation of MALAT1 and downregulation of miR-26a were detected in human posterior capsule opacification (PCO) attached LECs and the LECs obtained from patients with anterior polar cataracts by quantitative RT-PCR (qRT-PCR). Next, our results showed that TGF-β2 induces overexpression of EMT markers in primary HLECs via a MALAT1-dependent mechanism. The mechanism is that MALAT1 negatively regulates miR-26a and miR-26a directly targets Smad4 by luciferase reporter assays and RNA-binding protein immunoprecipitation assay. In summary, TGF-β2 induces MALAT1 overexpression, which in turn MALAT1 acts as a ceRNA targeting Smad4 by binding miR-26a and promotes the progression of EMT of LECs.

EGF-Mediated Overexpression of Myc Attenuates miR-26b by Recruiting HDAC3 to Induce Epithelial-Mesenchymal Transition of Lens Epithelial Cells.

The previous study has demonstrated that epidermal growth factor (EGF) and EGF receptor (EGFR) signaling plays a critical role in the development of posterior capsule opacification (PCO) through regulating lens epithelial cells (LECs) proliferation. Recent studies have suggested that the residual LECs undergo proliferation and migration, and epithelial-mesenchymal transition (EMT) is the important cause of PCO formation after cataract surgery. EMT of LECs is considered to be playing a central role in the pathogenesis of PCO. In the present study, we investigated whether and how EGF may regulate EMT of LECs. First, we demonstrated that EGF and EGFR signaling induces Myc overexpression in primary human lens epithelial cells (HLECs). In turn, Myc overexpression could inhibit miR-26b by recruitment of HDAC3. Consequently, the downregulated expression of miR-26b increased the expression of EZH2 in primary HLECs. Mechanistically, miR-26b directly controls EZH2 expression by targeting its 3′-UTR in HLECs by luciferase reporter assays. Finally, we demonstrated that EGF induces the expression of EMT markers in primary HLECs via a miR-26b-dependent mechanism. In summary, EGF activated Myc and Myc overexpression inhibited miR-26b by recruitment of HDAC3, which in turn induced the expression of EZH2 and promoted the progression of EMT in HLECs.

Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma

PurposeThe hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. MethodsBaseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. ResultsPrimary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. ConclusionsThe lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.

Role of Phospholipase D2/Phosphatidic Acid Signal Transduction in μ- and δ-Opioid Receptor Endocytosis

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.

Importance of calcium and potassium currents in human lens epithelial cells (hLEC) and the effect of the calcium channel blocker mibefradil

Background: To prevent posterior capsule opacification (PCO), we followed the hypothesis that calcium channel blockers (antagonists) interfere with integrin signaling and block cell adhesion in lens epithelial cells (LEC). In primary human LEC we found that the T channel antagonist mibefradil induces apoptosis which was accompanied with cell shape changes and loss of cell adhesion. Although T-type calcium channels are substantially present in membranes of freshly dispersed primary cultured hLEC and calcium currents are inhibited by mibefradil at concentrations of 10-8 M, the antiproliferative site of action of this drug remains unclear, since this feature is observed at concentrations 200-fold higher than that for calcium channel blockade. Methods: Epithelial cells of the human lens were dispersed by enzymatic treatment, recordings of membrane currents were performed using patch clamp technique in the whole cell configuration. Westernblot analysis was used for protein detection. Results: Total current elicited on depolarizing voltage steps from a holding potential of-80 mV was composed of inward (calcium) and outward (potassium) current. Outward current could be inhibited mostly by intracellular application of cesium ions. Currents in inward direction were activated fast (< 2 ms) and inactivated during the following 20 ms. They were characterized as calcium currents since the known calcium channel inhibitor nifedipine blocked these currents in a concentration-dependent manner. Using potassium in the pipette (145 mM) as main charge carrier, additionally a noninactivating potassium current and a voltage- and time-dependent potassium current which slowly inactivated (Kv) were observed (control). Adding mibefradil in concentrations from 10-6 M - 10-5 M to the bath solution, the inwardly directed and the non-inactivating current component were inhibited concentration-dependent. The Kv component was affected in a similar way, however, this component showed an increased inactivation behavior after application of mibefradil in the named concentrations. Conclusion: The total effects of mibefradil are significant for the calcium homeostasis since calcium current itself is inhibited but, moreover, the membrane is permanently depolarized up to 20 mV by the drug. Both effects may contribute to the observed reduced cell adhesion during mibefradil treatment. (Less)

Specific Activation of the Glucocorticoid Receptor and Modulation of Signal Transduction Pathways in Human Lens Epithelial Cells

Prolonged use of glucocorticoids (GCs) can lead to cataract formation. Lens GC responses have been difficult to elucidate. A previous study showed the presence of the glucocorticoid receptor (GR) in immortalized and primary human lens epithelial cells (hLECs) and GC-induced changes in gene expression. This study demonstrates specific GR activation and identifies the biological effect of GC-induced changes in gene expression in hLECs. HLE B-3 (B-3) and primary cultures of hLECs were transfected with pGRE.Luc and treated with or without dexamethasone (Dex), RU-486, spironolactone, or vehicle. mRNA and protein expression were examined by real-time PCR and Western blot analysis, respectively. Cell proliferation and apoptosis were examined by WST-1 and flow cytometry, respectively. Dex treatment of B-3 and primary cultures demonstrated specific GR, but not mineralocorticoid receptor (MR), activation and phosphorylation. Pathway analysis revealed GC-induced changes in expression of MAPK regulators. Increased expression of GILZ mRNA and MKP-1 mRNA and protein was observed in immortalized and donor hLECs. This corresponded with a decrease in the phosphorylated forms of RAF, ERK, p38, and AKT, but not in JNK. No net change in LEC proliferation or apoptosis was observed with Dex treatment. GC treatment of hLECs activates the GR to modulate the expression of MAPK and PI3K/AKT regulators. This is the first demonstration of GC signaling in hLECs. GCs, MAPK, and PI3K/AKT are involved in cell processes implicated in steroid-induced cataractogenesis. The absence of a net change in cell activity with acute steroid treatment is consistent with the possibility that chronic treatment leads to prolonged modulation of these pathways and steroid-induced cataract.

Suppression of Human Lens Epithelial Cell Proliferation by Proteasome Inhibition, a Potential Defense against Posterior Capsular Opacification

Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway. HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-beta2 (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 muM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition. TGF-beta2 suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-beta2 was blocked by adding FGF-2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF-beta2, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 protein. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132. Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-beta2, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.

Induction of apoptosis by the calcium antagonist mibefradil correlates with depolarization of the membrane potential and decreased integrin expression in human lens epithelial cells.

Posterior capsule opacification is still the major complication in cataract surgery and is caused by migration and proliferation of residual lens epithelial cells. The challenge of a suitable therapy to inhibit capsule opacification is to specifically interfere with cellular mechanisms. Our approach using the T-calcium channel antagonist mibefradil is based on the hypothesis that this drug inhibits the signaling pathways mediated by cell adhesion. The influence of mibefradil dihydrochloride was investigated on primary human lens epithelial cells (hLEC) from cataract surgery and on the human lens cell line HLE-B3. Apoptosis was quantitatively analyzed by flow cytometry (% increase of the sub-G1 peak), and verified by confocal microscopy (annexin V-biotin, TUNEL reaction). The membrane potential was detected by a membrane potential-sensitive dye. Integrin expression and proliferation were measured by flow cytometry. T-calcium channels in hLEC were verified by the whole-cell configuration of the patch-clamp technique. Mibefradil induced apoptosis in hLEC. Early signs of apoptosis were observed after only 4 h of incubation with mibefradil, accompanied by a significantly reduced cell area. Apoptosis correlated with inhibited integrin expression, reduced proliferation and the depolarization of the membrane potential. We could identify calcium channels of the T-type in our primary hLEC. We suggest that depolarization of the membrane potential and the inhibition of integrin expression leads to the loss of cell adhesion, which is the reason for the induction of apoptosis. Thus, mibefradil seems to be a suitable drug to prevent cell adhesion, migration and proliferation.

Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses.

To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses. Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy. Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses. Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.