Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

Abstract

␣A-and ␣B-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of ␣A-and ␣B-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of ␣A-and ␣B-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of ␣A-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of ␣B-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor ␣. To further study the relative protective activities of ␣A-and ␣B-crystallins, we created a cell line derived from ␣A؊/؊␣B؊/؊ double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line ␣A␣BKO1 derived from ␣A/␣B double knockout cells was transfected with ␣A-or ␣B-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either ␣A-crystallin or ␣B-crystallin were isolated. The ability of ␣A-or ␣B-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine-or UVA-treated cells. The results indicate that the anti-apoptotic activity of ␣A-crystallin was two to threefold higher than that of ␣B-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of ␣A-and ␣B-crystallin. Since the expression of ␣A-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

“…[39][40][41][42][43][44][45][46][62][63][64][65][66][67][68][69][70][71][72][73][74] One of the most important functions is the ability to protect cell from induced apoptosis. 39-46 a-Crystallin and Hsp27 are closely related family members and protect cells from apoptosis induced by a large numbers of stress factors.…”

“…39-46 a-Crystallin and Hsp27 are closely related family members and protect cells from apoptosis induced by a large numbers of stress factors. [39][40][41][42][43][44][45][46] Both of them prevent stress-induced apoptosis at multiple signaling steps. However, the exact mechanism or regulating targets seem to be different.…”

“…An early study 43 has suggested that aA-crystallin is more competent than aB-crystallin in preventing apoptosis induced by staurosporine, TNF-a and UVA. In the present study, we have compared the antiapoptotic ability of the two crystallins in three different cell lines: the human lens epithelial cell line (HLE) which contains virtually undetectable endogenous crystallins; the human retina pigment epithelial cells (ARPE-19) and the rat embryonic myocardium cell line (H9c2), both of which expresses endogenous aB-crystallin.…”

“…Its proapoptotic ability can be inhibited by Bcl-2 and Bcl-X L . 38 Another important group of apoptosis regulators are heatshock proteins that include alpha-crystallins (a-crystallins), [39][40][41][42][43][44][45][46] Hsp27, [39][40][47][48][49][50][51][52] Hsp70, [53][54][55][56][57] Hsp90 58 and Hsp60. [59][60] While Hsp60 appears to enhance apoptosis by promoting maturation of procaspase-3, 59,60 the majority of these factors seems to protect cells from induced apoptosis through different mechanisms.…”

“…68,69 As antiapoptotic regulators, acrystallins are initially shown to protect cells from thermal, 70 osmotic 71 and oxidative insult. 72 More recently, a-crystallins have been shown to prevent induced apoptosis by various factors including staurosporine, [39][40]43 TNF, 39,43 UVA irradiation, 43 okadaic acid 44 and hydrogen peroxide. 45 Regarding the antiapoptotic mechanism, recent studies from our laboratory and another group have demonstrated that aBcrystallin can directly interact with the precursors of caspase-3 to suppress its activation.…”