Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

Purpose: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells.

Methods: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA.

Lenticular cytoprotection, part 2: link between glycogen synthase kinase-3β, epithelial to mesenchymal transition, and mitochondrial depolarization.

Purpose: The inhibition of GSK-3β blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3β, as part of a multifactorial complex, also regulates nuclear levels of β-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between β-catenin's influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear β-catenin as an inducer of epithelial to mesenchymal transition (EMT).

Lenticular mitoprotection. Part B: GSK-3β and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Purpose: Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3β) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3β, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3β influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.

Purpose: Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection).

Lenticular cytoprotection. Part 1: the role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia.

Purpose: The prosurvival signaling cascades that mediate the unique ability of human lens epithelial cells to survive in their naturally hypoxic environment are not well defined. Hypoxia induces the synthesis of the hypoxia inducible factor HIF-1α that in turn, plays a crucial role in modulating a downstream survival scheme, where vascular endothelial growth factor (VEGF) also plays a major role. To date, no published reports in the lens literature attest to the expression and functionality of HIF-2α and the role it might play in regulating VEGF expression.

Characterization and functional expression of the natriuretic peptide system in human lens epithelial cells.

Purpose: The family of natriuretic peptides (NPs); atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three associated receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C) has never been documented in human lens epithelial cells. The study described herein was designed to demonstrate both expression and functionality of components of the natriuretic peptides and natriuretic peptide receptors in the human lens epithelial cell line, HLE-B3 and in normal human lens epithelial cell cultures (nHLE).

Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) along with confirmation by DNA sequencing and real-time quantitative RT-PCR was used to identify and demonstrate expression of mRNA for the natriuretic peptide family.

Regulation of mitochondrial respiratory chain biogenesis by estrogens/estrogen receptors and physiological, pathological and pharmacological implications.

There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17beta-estradiol (E(2)) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E(2)-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERalpha and ERbeta and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anti-cancer drug resistance in human breast cancer cells, neuroprotection for Alzheimer's disease and Parkinson's disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area.

Mitochondrial superoxide dismutase activation with 17 beta-estradiol-treated human lens epithelial cells.

Purpose: 17 beta-estradiol (17beta-E(2)) protects human lens epithelial cells against oxidative stress by preserving mitochondrial function in part via the non-genomic rapid activation of prosurvival signal transduction pathways. The study described herein examined whether 17beta-E(2) also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible parallel mechanism by which 17beta-E(2) protects against oxidative stress.

Methods: Virally-transformed human lens epithelial cells (HLE-B3) were pre-incubated with 17beta-E(2), and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h.

RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells.

17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway.

Estradiol attenuates mitochondrial depolarization in polyol-stressed lens epithelial cells.

Purpose: This study examined the state of mitochondrial physiology subsequent to exposing lens epithelium to high ambient galactose (Gal), which upon conversion to galactitol (GalOH) and resultant intracellular accumulation thereof, leads to profound destabilization of mitochondrial membrane potential (Deltapsim). Further, we determined whether the aldose reductase (AR) inhibitor, Sorbinil, or estrogen (17beta-E2, and its isomer, 17alpha-E2, which exhibits marginal binding affinity for estrogen receptor), administered prior to and concomitant with Gal exposure might prevent or delay mitochondrial membrane depolarization.

Methods: Secondary cultures of bovine lens epithelial cells (BLECs), as well as a virally-transformed human lens epithelial cell line (HLE-B3), were maintained in 40 mM galactose (Gal) for up to seven days in the presence and absence of Sorbinil, 17beta-E2 or 17alpha-E2.

Differential expression and comparative subcellular localization of estrogen receptor beta isoforms in virally transformed and normal cultured human lens epithelial cells.

A number of variants of the wild-type (wt) estrogen receptor beta (ERbeta-1) coexist in a wide range of tissues. In the human these include, together with others, the expression of several isoforms (ERbeta-2-ERbeta-5) due to alternative splicing of exons encoding the carboxy terminus. In this study, we determined whether virally transformed cell cultures of human lens epithelial cells (HLE-B3) express both full length (or wt) and variant isoforms of ERbeta in comparison to normal secondary cultures of human lens epithelial cells (nHLE) and furthermore, identify the subcellular localization of the wtERbeta-1 and ERbeta isoform variants in HLE-B3 and nHLE cells, as well as from human breast adenocarcinoma cells (MCF-7) which provided a positive control.

17beta-estradiol stimulates MAPK signaling pathway in human lens epithelial cell cultures preventing collapse of mitochondrial membrane potential during acute oxidative stress.

17beta-estradiol (17beta-E2) protects against H2O2-mediated depletion of intracellular ATP and lessens the degree of depolarization of mitochondrial membrane potential (DeltaPsi(m)) in cultured lens epithelial cells consequential to oxidative insult. We now report that 17beta-E2 acts as a positive regulator of the survival signal transduction pathway, MAPK which, in turn, acts to stabilize DeltaPsi(m) in effect, attenuating the extent of depolarization of mitochondrial membrane potential in the face of acute oxidative stress. The SV-40 viral transformed human cell line, HLE-B3 was treated with 17beta-E2 over a time course of 60 min and phosphorylation of ERK1/2 was analyzed by Western blot.

A putative mitochondrial mechanism for antioxidative cytoprotection by 17beta-estradiol.

It has been demonstrated that estrogens are potent antioxidants and protect against H2O2-mediated depletion of intracellular ATP in human lens epithelial cells (HLE-B3) [Invest. Ophthalmol. Vis.

Subcellular distribution of native estrogen receptor alpha and beta subtypes in cultured human lens epithelial cells.

The expression and intracellular localization of the estrogen receptor (ER) subtypes, ERalpha and ERbeta were examined in cultured human lens epithelial cells (HLE-B3). ERalpha and ERbeta mRNA expression was evaluated by coupled reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Subcellular localization of ERalpha and ERbeta was determined on formaldehyde-fixed, Saponin-permeabilized cells using conventional immunofluorescence techniques, as well as immunodetection of differential cellular components after subjecting the cultured cells to fractionation by sucrose gradient centrifugation.

Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential, ATP, and cell viability.

Purpose: Epidemiologic studies demonstrate a higher incidence of cataracts in estrogen-deprived postmenopausal women, but the mechanism for the increased risk of cataracts is unclear. An elevated level of H(2)O(2) in aqueous humor and whole lenses has been associated with cataractogenesis. In the present study, for the first time, the protective effect of estrogens against oxidative stress were tested in cultured human lens epithelial cells (HLECs).

Osmoregulatory alterations in taurine uptake by cultured human and bovine lens epithelial cells.

Purpose: Comparative assessment of cultured human lens epithelial cells (HLECs) and bovine lens epithelial cells (BLECs) established the nature of the relationship between taurine-concentrating capability and intracellular polyol accumulation or extracellular hypertonicity.

Methods: The kinetic characteristics of active taurine accumulation based on the measurement of in vitro [3H]-taurine uptake were resolved by side-to-side review of cultured HLECs and BLECs pre-exposed to either galactose-supplemented medium or extracellular hypertonicity. Competitive RT-PCR was used to appraise variation in taurine transporter (TauT) mRNA abundance from cells maintained in hyperosmotic medium over a 72-hour exposure period.