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

Abstract

. 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. We found that E 2 rapidly increased both the phosphorylation of ERK2 and Ser 112 in BAD. Ser 112 is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E 2-induced phosphorylation of Ser 112 . Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism. 17␤-estradiol; mitochondrial membrane potential; mitochondrial permeability transition OXIDATIVE STRESS CAUSES PROFOUND INJURY to a diverse number of intracellular macromolecules in eukaryotes, including lipid peroxidation, protein alteration, breakage of covalent bonds of carbohydrates, and cleavage of DNA strands. Mitochondria are particularly susceptible to oxidative damage with consequent depolarization of mitochondrial membrane potential (8,10,24). Hyperoxic oxygen uncouples mitochondrial electron transport in human lens epithelial cells (HLE-B3) cells. This uncoupling of electron transport increases the levels of reactive oxygen species (ROS; Ref. 22) and decreases cellular ATP production. Similarly, hydrogen peroxide (H 2 O 2 ) directly collapses mitochondrial membrane potential (⌬⌿ m ) in a variety of cell types, causing decreases and increases in ATP and ROS production, respectively (12).Pretreatment of cells with estrogen prevents most of the mitochondrial changes elicited by oxidative stress. Estradiol blocks membrane oxidation at physiological concentrations (17). Estrogen treatment reduces lipid peroxidation induced by glutamate and attenuates the increase in intracellular peroxide induced by bolus H 2 O 2 addition (16) or by mitochondrial toxins (41). Mitochondria play a central role in the generation of biological forms of energy and also in the production of ROS. The damage observed in mitochondria from disease and/or experimental insults such as H 2 O 2 lead to deficiency in ATP production, as well as a concomitant increase in production of ROS, overwhelming cellular antioxidant defense systems. Under conditions of oxidative stress, mitochondria underg...

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

“…In agreement, estrogen has previously been reported to increase MnSOD activity levels without affecting the protein levels of MnSOD in mitochondria of rat pheochromocytoma cells (PC-12 cells) [367] and MCF-7 breast cancer cells [54]. Within the same timeframe that estradiol increases MnSOD activity, estradiol also initiates a signaling cascade culminating in the activation of the Ras/Raf/ERK (MAPK) and PKA pathways in cultured HLE-B3 cells [366]. …”

“…The primary focus of the Cammarata laboratory has been to demonstrate that 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[59, 217, 366]. Newly obtained information, derived from these recent studies, necessitates a reevaluation of the past interpretation of the cytoprotective scheme.…”

“…Transfection of HLE-B3 cells with ERK2-specific siRNA enhanced mitochondrial membrane depolarization subsequent to peroxide insult whereas Bad siRNA-treated and mock-transfected cells did not. The implication is that ERK2, but not Bad, plays a pivotal role in the regulation of mitochondrial membrane potential in human lens epithelial cells[366]. Estrogen treatment does, however, continue to offer some protection against mitochondrial membrane depolarization, despite ERK2 suppression.…”

“…ERK2, and more specifically pERK2, represents one pathway of the estrogen-mediated mitochondrial protection pathway. Alternatively, estrogen may, in part, supersede the necessity for ERK activation and provide a measure of protection via an ERK-independent route[366]. …”

The influence of gender and sexual hormones on incidence and outcome of chronic kidney disease

“…This cellular pathway could explain why skeletal muscle cells are stabilized against oxidative damage by estrogens [52] and why degenerative diseases of muscle cells are associated with a decrease of estrogen levels [53]. The stabilization of mitochondrial function has also been shown in ocular lens epithelium, where estrogen was found to reduce destabilization of mitochondrial membrane potential by oxidative stress [54]. In addition to these genomic ER effects, 'nonclassical' actions via membrane-associated ER are also able to activate cytoplasmic signaling pathways in a G-proteincoupled manner.…”