Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 5, 1 September 2012, Pages 1111-1122
Free Radical Biology and Medicine

Original Contribution
Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

https://doi.org/10.1016/j.freeradbiomed.2012.06.042 Get rights and content

Abstract

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (–/–) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.

Highlights

▸ ER stress in retinal pigment epithelium (RPE) leads to mitochondrial dysfunction. ▸ ER stress in RPE results in apoptosis from activation of caspases−4 and −3. ▸ ER stress-mediated apoptosis in RPE involves PERK-eIf2α, ATF6, and CHOP. ▸ αB Crystallin deficiency in RPE augments ER stress-induced apoptosis. ▸ αB Crystallin overexpression protects RPE from ER stress-induced apoptosis.

Introduction

Age-related macular degeneration (AMD), a progressive degenerative retinal disease, is the leading cause of blindness among the elderly in developed countries. Retinal pigment epithelium (RPE) cells offer essential nutritional and metabolic support for light-sensitive photoreceptors, and are a major pathologic target in the early and late stages of AMD [1]. Multiple cellular mechanisms are involved in the dysfunction and death of RPE cells in AMD, including accumulation of toxic metabolites, oxidative stress, and inflammation [1], [2]. Recent studies suggest that endoplasmic reticulum (ER) stress also participates in the pathogenesis of RPE dysfunction in retinal degenerative disorders including AMD [3], [4].

Accumulation of unfolded proteins in the ER lumen results in “ER stress” and initiates a complex cellular response known as the unfolded protein response (UPR) [5]. This response is mediated through three ER transmembrane receptors: PRK (RNA-dependent protein kinase)-like ER/pancreatic eukaryotic translation initiation factor 2 subunit (eIF2) kinase (PERK/PEK), activating transcription factor-6 (ATF6), and the inositol-requiring enzyme 1(IRE1). Once UPR is triggered, cells first establish adaptive responses such as induction of ER chaperone GRP78 or global inhibition of protein synthesis. However, if this transcriptional program fails to reestablish ER homeostasis, signaling switches to a proapoptotic pathway [6].

The specific mechanisms involved in ER stress-induced apoptosis remain to be fully elucidated. The available evidence suggests that transcriptional induction of CHOP is critical in ER stress-induced apoptosis [7]. Overexpression of CHOP can lead to cell cycle arrest and apoptosis, while CHOP (–/–) cells attenuated apoptosis in response to ER stress [7]. Activation of caspase cascades also occurs in ER stress. In rodents, earlier studies proposed that caspase 12 played a role in ER stress-induced apoptosis [6], [8], even though deletion of caspase 12 provides only partial protection against cell death [9]. However, a caspase 12-like protein in human cells contains a polymorphism that results in a truncated nonfunctional protein [10]. Thus, recent studies have focused attention on caspase 4, which has been proposed to fulfill the function of caspase 12 in humans [11]. Caspase-dependent pathways of ER stress-induced apoptosis that are independent of caspase 4/12 have also been identified [12].

Mitochondria are recognized as the central regulator of apoptotic cell death and ER-mitochondrial cross talk may mediate stress signals between these compartments [13]. Indeed, mitochondrial changes including loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 9 and caspase 3 have been observed in ER stress [14]. A link between ER stress and reactive oxygen species (ROS), decrease in glutathione (GSH), and increase in calcium influx in the mitochondria has also been shown [15], [16], [17]. It has also been reported that CHOP, the ER stress-induced transcription factor, not only down regulates Bcl-2 expression but also leads to translocation of Bax from cytosol to mitochondria [7], [18], [19]. The activation and mitochondrial localization of Bax [20], [21] and Apaf 1 (apoptotic protease-activating factor 1), which is required in postmitochondrial apoptotic cascades, have been identified to contribute to mitochondrial impairment in ER stress-induced apoptosis [22]. Thus, mitochondria are clearly linked to the development of ER stress-induced apoptosis.

Recently, the small heat shock protein αB crystallin has been identified as an important regulator of mitochondrial apoptosis; it inhibits oxidant-induced apoptosis in RPE and progression of retinal degeneration in animal models [23], [24], [25]. Expression of αB crystallin has also been linked to AMD, where its increased expression has been suggested to be a biomarker for the disease [26]. Expression of αB crystallin in RPE and its secretion from the apical surface of human polarized RPE mediates neuroprotection to adjacent cells [23]. αB crystallin has been localized to several intracellular compartments including the mitochondria [24], but little is known about its effect on ER stress and mitochondrial dysfunction. In the present study, we evaluate the role of αB crystallin in ER stress-mediated apoptosis in RPE, and demonstrate that αB crystallin provides critical protection of mitochondrial function in this process.

Section snippets

Chemicals and materials

Tunicamycin (TM) and AEBSF were obtained from Sigma Aldrich (St. Louis, MO). Salubrinal was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Caspase 4 inhibitor was obtained from BioVision (Mountain View, CA).

The αB crystallin knockout mice with 129S6/SvEvTac background were obtained from the National Eye Institute (courtesy of Dr. Eric Wawrousek, Ph.D.), while 129S6/SvEvTac control mice were purchased from Taconic Farms (Germantown, NY). Isolation, culture, and characterization of

Results

Pilot studies were conducted with several proapoptotic agents that specifically induce ER stress. They included tunicamycin (an inhibitor of N-linked glycosylation), brefeldin (BFA, an inhibitor of ER-Golgi transport), and thapsigargin (TG, an inhibitor of ER Ca2+ uptake). These studies showed that in the absence of αB crystallin, all three ER stressors induced cell death when compared to wild-type RPE cells (data not shown). We chose TM to induce ER stress in RPE cells since TM has been used

Discussion

Our data show that ER stress activates caspase 4 and the cell death that results also involves several mitochondrial apoptotic events. Further, we also show that deficiency of αB crystallin renders RPE susceptible to ER stress-induced cell death while overexpression results in protection. We have recently reported that deficiency of αB crystallin protects against development of choroidal neovascularization (CNV) in mice [27]. Here we show that αB crystallin protects RPE cells from both

Acknowledgments

This work was supported in part by Grant EY01545 (SJR, DRH) and by core Grant EY03040; the Arnold and Mabel Beckman Foundation; an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness Inc., New York, NY. We thank Jennifer Yaung, Ph.D. for assistance in preliminary experiments and Eric A. Barron and Ernesto Barron for expert technical help.

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